Tomato fruit having increased firmness

ABSTRACT

This invention relates to a tomato fruit with significantly increased firmness due to the presence of at least one genetic element (or quantitative trait loci; QTLs) in the cultivated plant producing said tomato fruit, compared to fruit from a control tomato plant which does not have said genetic element(s). A cultivated tomato plant producing tomato fruit with significantly increased fruit firmness and a method for detecting QTLs linked to significantly increased fruit firmness are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/500,149(now U.S. Pat. No. 8,987,549 B2), which claim priority under 35 U.S.C. §371 from PCT Application No. PCT/EP2010/065575, filed Oct. 15, 2010,which claims the benefit of European Patent Application No. 09174072.0,filed Oct. 26, 2009, the disclosures of which are incorporated byreference herein in their entirety.

STATEMENT REGARDING ELECTRONIC SUBMISSION OF A SEQUENCE LISTING

A Sequence Listing in ASCII test format, submitted under 37 C.F.R.1.821, entitled “72652_ST25.txt”, 111 kilobytes in size, generated onFeb. 12, 2015 and filed via EFS-Web is provided in lieu of a paper copy.This sequence listing is hereby incorporated by reference into thespecification for its disclosure.

INTRODUCTION

For all fruit products, fruit firmness which is fit for purpose isessential to meet and possibly exceed consumer expectations. Consumerswill reject products with an unacceptable fruit firmness, even thoughother quality attributes such as flavour and colour are good orexcellent. Additionally, the supply chain requires an appropriate levelof fruit firmness for the effective delivery of high quality fruit toretail outlets. Improving the quality of the raw materials will alsoencourage the development of healthier diets.

Tomato is the model system for studying fruit ripening due to extensivegenetic and genomic resources. Fruit firmness in tomato is determined bya number of factors including cell wall structure, turgor and cuticleproperties. Fruit firmness is a complex trait and depends on the actionof many genes. This has made it difficult to dissect the eventsdetermining changes in fruit firmness by focusing on changes in cellwall degrading enzymes.

Harvesting tomato fruit when ripening has set in would make maturitydetermination easier as it would be based on visible peel color andwould assure full quality development. After harvest, ripening continuesand softening advances, increasing the susceptibility of the fruit tohandling damage and limiting the marketing period. Slowing down theripening and softening stages would allow harvesting, transport andstorage of partially ripe but firm fruit (T. Chanthasombath et al,2008).

Ripening mutants in tomato such as Colourless non-ripening and ripeninginhibitor have yielded important insights into an emerging geneticframework which regulates ripening and modulates fruit firmness(Thompson et al, 1999; Vrebalov et al, 2002; Eriksson et al, 2004;Manning et al, 2006). Delaying ripening and softening may be achieved byemploying modified atmosphere packaging (MAP) which has been extensivelystudied as a simple and cheap method of prolonging shelf life of manyfruits and vegetables including tomato (Batu & Thompson, 1998, Exama etal, 1993, Geeson et al, 1985), however it increases the cost ofpackaging and handling of fruits. Existing methods to enhance fruitfirmness in conventional plant breeding programs rely on screening fruitfirmness differences in fruit harvested from mature plants. Anyidentification of enhanced fruit firmness in this scenario will largelybe down to chance. Currently it is not financially viable or efficientto breed for enhanced fruit firmness due to the cost and complexity ofgrowing and phenotyping large numbers of plants.

To bridge the gap between the emerging model for the regulation of fruitripening and a full knowledge of the components involved in controllingfruit firmness will require additional strategies to those based oneither targeting genes for known cell wall-related proteins orinvestigating pleiotropic ripening mutants. Fruit firmness is aquantitative trait involving many genes and yet the identity of themajority of these genes remains elusive.

Wild tomato species offer a rich and largely unexplored source of newgenetic variation for breeders. Tanksley and Zamir (Frary et al, 2000;Fridman et al, 2004) have demonstrated that this source of geneticdiversity can be used to understand the molecular basis of importantfruit quality traits and provide new material for breeding.

It is thus apparent from the prior art that there is a need to modifytomato fruit firmness and to delay the softening of tomato fruits inparticular so that the fruits stay on the vine for a longer period oftime. The fruits would then remain firmer while accumulating moreflavour and taste components and thus exhibit a final better tastecompared to fruits that have been harvested earlier and ripenedartificially with gas for example. Such increased firmness of the fruitwould allow a longer period of time for harvesting since the fruitscould be left on the vine without the associated softening which wouldbe detrimental for the steps of handling, packing and delivery to thesupermarket shelves.

SUMMARY OF THE INVENTION

An investigation has been conducted on the distribution of quantitativetrait loci (QTL) for tomato fruit mechanical properties on the publiclyavailable ‘Zamir’ lines (Eshed and Zamir, 1994). These are a set of 76Solanum pennellii introgression lines (ILs) generated in a Solanumlycopersicum background (cv. M82). These ILs partition the genetic mapinto 107 bins that are defined by singular or overlapping S. pennelliisegments. This targeted population, which allows ‘Mendelisation’ of QTLpermits mapping and resolution of traits while using a manageableexperimental system to take account of environmental effects.

The inventors have defined genetic elements (or QTL) which aresurprisingly linked to significantly increased fruit firmness in tomato.The present invention therefore relates in a first aspect to a tomatofruit with significantly increased fruit firmness at the harvestingstage, preferably at the mature green stage, or at the breaker plus 7days stage, wherein said increased fruit firmness is linked to saidgenetic elements in the cultivated tomato plant producing said tomatofruit, wherein said firmness is from between 1.2 to 2 times greater or,alternatively, 1.2 to 1.5 times greater than that of fruit from acontrol tomato plant which does not have these genetic elements.Introgression lines IL2-3 and IL2-4 (deposited under accession numbersLA4038 and LA4039 respectively), and which bear these genetic elements,showed a significant increase in fruit firmness compared to an M82parental control with the most pronounced effects observed in IL2-3.There is a dominant effect of these genetic elements in the F1generation and the trait is most apparent in ripe tomato fruit.

There is also provided a cultivated tomato plant which produces tomatofruit with increased firmness as described above, wherein said plant canbe characterised by a) the at least one genetic element is linked to atleast one DNA marker selected from the group consisting of NT3853,NT3907, TG14, HOX7A, CT277, HB2600, TG353, Lm0127, Lm1650, LE5100 andLE5200 and/or b) the at least one genetic element is complementary tothe corresponding genetic element in S. pennellii lines IL2-3 and IL2-4,wherein the said genetic element in IL2-3 and IL2-4 is linked to atleast one DNA marker selected from the said group in (a).

There is also provided a cultivated tomato plant wherein the at leastone genetic element is one or more QTL selected from a) QTL1 linked toat least one of the DNA markers NT3853, NT3907 and TG14; b) QTL2 linkedto at least one of the DNA markers HOX7A and CT277; c) QTL3 is linked toat least one of the DNA markers HB2600, TG353; d) QTL4 linked to atleast one of the DNA markers Lm0127 and Lm1650; e) QTL5 linked to atleast one of the DNA markers LE5100 and LE5200, and combinations ofthese QTLs thereof.

There is also provided a cultivated tomato plant as described abovewherein the QTL is QTL1 linked to at least one of the DNA markersNT3853, NT3907 and TG14; which is present only in the inner pericarp.

There is also provided a cultivated tomato plant as described abovewherein said plant is an inbred, a diploid or a hybrid. In a specificembodiment, the plant is male sterile.

There is also provided a tomato seed which produces a cultivated tomatoplant of the present invention.

There is also provided a plant part of a cultivated tomato plant asdescribed herein. In a specific embodiment there is provided plantmaterial obtainable from a plant part of a cultivated tomato plant asdescribed herein.

The above identified DNA markers represent a rapid assay and researchtool to select for cultivated tomato plants containing a defined geneticregion. These DNA markers yield the potential to manipulate a very smalland well defined genetic region which is linked to increased fruitfirmness. This has the advantage of allowing the screening of largenumbers (1000's) of plants at a very early stage of development fordesirable combinations of DNA markers and will allow the enhancement offruit firmness in commercial varieties of tomato.

There is also provided a method for detecting a QTL linked tosignificantly increased fruit firmness in fruit from a cultivated tomatoplant as described above compared to a control tomato plant comprisingthe steps of a) crossing a donor tomato plant with a recipient tomatoplant to provide offspring plants, b) quantitatively determining thefirmness in the fruit of said offspring plants c) establishing a geneticlinkage map that links the observed increased fruit firmness to thepresence of at least one DNA marker from said donor plant in saidoffspring plants and d) assigning to a QTL the DNA markers on said mapthat are linked to significantly increased fruit firmness.

Preferably said donor plant has fruit with a significantly increasedfruit firmness compared to said recipient plant.

Preferably, the donor plant is S. pennellii and the recipient plant isS. lycopersicum. The fruit firmness range in the offspring plants is 1.2to 2 times greater, or alternatively 1.2 to 1.5 times greater, than thatof fruit produced from a control tomato plant at the harvesting stage.Preferably, the harvesting stage is the mature green stage. Preferablythe at least one DNA marker is selected from NT3853, NT3907, TG14,HOX7A, CT277, HB2600, TG353, Lm0127, Lm1650, LE5100 and LE5200.Preferably said QTL is one or more of a) QTL1 linked to at least one ofthe DNA markers NT3853, NT3907 and TG14; or b) QTL2 linked to at leastone of the DNA markers HOX7A and CT277; c) QTL3 linked to at least oneof the DNA markers HB2600, TG353; d) QTL4 linked to at least one of theDNA markers Lm0127 and Lm1650; or e) QTL5 linked to at least one of theDNA markers LE5100 and LE5200.

In one embodiment, said QTL is QTL1 linked to at least one of the DNAmarkers NT3853, NT3907 and TG14; which is present only in the innerpericarp.

There is also provided a QTL responsible for increased fruit firmness infruit of a cultivated tomato plant detected by a method as hereindescribed.

In one embodiment, said QTL is located on the long arm of chromosome 2.

In one embodiment there is provided a QTL as herein described linked toat least one DNA marker selected from the group consisting of NT3853,NT3907, TG14, HOX7A, CT277, HB2600, TG353, Lm0127, Lm1650, LE5100 andLE5200.

In one embodiment there is provided a QTL as herein described whereinsaid QTL is one or more of a) QTL1 linked to at least one of the DNAmarkers NT3853, NT3907 and TG14; b) QTL2 linked to at least one of theDNA markers HOX7A and CT277; c) QTL3 linked to at least one of the DNAmarkers HB2600, TG353; d) QTL4 linked to at least one of the DNA markersLm0127 and Lm1650; or e) QTL5 linked to at least one of the DNA markersLE5100 and LE5200.

In one embodiment there is provided a QTL as herein described whereinsaid QTL is QTL1 linked to at least one of the DNA markers NT3853,NT3907 and TG14; which is present only in the inner pericarp.

There is also provided an isolated DNA sample obtained from a tomatoplant comprising a QTL as herein described.

There is also provided a method of producing a tomato plant whichprovides fruit with increased fruit firmness as herein described.

There is also provided a method of producing an offspring cultivatedtomato plant which provides fruit with increased fruit firmnesscomprising the steps of performing a method for detecting a QTL linkedto increased fruit firmness, and transferring a nucleic acid comprisingat least one QTL thus detected, from a donor tomato plant to a recipienttomato plant, wherein said increased fruit firmness is measured in fruitfrom an offspring cultivated tomato plant compared to fruit from acontrol tomato plant. The transfer of nucleic acid can be performed byany of several methods known in the art e.g. transformation, byprotoplast fusion, by a doubled haploid technique or by embryo rescue.

Preferably, the fruit firmness range in the offspring tomato plant isgreater than fruit from a control tomato plant at the mature greenstage, or alternatively the breaker plus 7 days stage. The fruitfirmness range could be 1.2 to 2 times greater or, alternatively, 1.2 to1.5 times greater. Preferably, the donor plant is S. pennellii and therecipient plant is S. lycopersicum. Preferably, said QTL is one or moreof a) QTL1 linked to at least one of the DNA markers NT3853, NT3907 andTG14; or b) QTL2 linked to at least one of the DNA markers HOX7A andCT277; or c) QTL3 linked to at least one of the DNA markers HB2600,TG353; or d) QTL4 linked to at least one of the DNA markers Lm0127 andLm1650; or e) QTL5 linked to at least one of the DNA markers LE5100 andLE5200.

In one embodiment said QTL is QTL1 linked to at least one of the DNAmarkers NT3853, NT3907 and TG14; which is present only in the innerpericarp.

There is also provided a tomato plant, or part thereof, obtainable by amethod as described herein.

There is also provided a cultivated tomato plant comprising a QTLresponsible for increased fruit firmness as described herein.

There is also provided a hybrid tomato plant, or part thereof,obtainable by crossing a tomato plant as described herein with a tomatoplant that exhibits commercially desirable characteristics.

There is also provided tomato seed produced by growing a tomato plant asdescribed herein.

There is also provided tomato seed produced by crossing a tomato plantas described herein with a plant having desirable phenotypic traits toobtain a plant that has significantly increased fruit firmness comparedto a control plant.

There is also provided the use of a QTL as described herein for theproduction of a tomato plant having increased fruit firmness compared toa control plant.

There is also provided the use of a tomato plant for expanding theharvesting slot of tomato fruit and/or for use in the fresh cut marketor for food processing.

There is also provided processed food made from a tomato plantcomprising the at least one QTL as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Representation of (A) IL2-3 (B) IL2-4 and (C) QTL-NIL 301introgressions. Proximal and distal markers are used to delimitintrogressed regions (shown in black) from M82 regions (shown in grey).

FIG. 2 Mechanical measurements on (Panel A) the outer pericarp and(Panel B) the inner pericarp of IL2-3F1 lines reveals enhanced fruitfirmness up to and including the breaker+7 day stage in comparison withthe M82 controls. Each graph shows the maximum load (N) on the Y-axisand fruit developmental stage on the X-axis. Line M82 is represented bya solid line. Line IL2-3F1 is represented by a dotted line. Fruitdevelopment stages: MG, mature green stage; B, breaker stage; B+3,breaker stage plus 3 days; B+5, breaker stage plus 5 days; B+7, breakerstage plus 7 days; B+10, breaker stage plus 10 days.

FIG. 3 Genotyping of randomly selected lines of (Panel A) IL 2-3 F2 and(Panel B) IL 2-4 F3 to show distribution of recombination events withrespect to Syngenta SSR and published RFLP and PCR-based markers. Key:A=M82; B=S. pennellii; H=heterozygote; *=missing data. The SSR markersare shown in the left hand column. The selected lines are shown on thetop row.

FIG. 4. Location of QTL for increased fruit firmness on tomatochromosome 2 as determined by measuring fruit firmness in (Panel A) theouter pericarp and (Panel B) the inner pericarp. QTLs are shown as blacksolid bars. For the outer pericarp, from left to right the QTLscorrespond to QTL2, QTL4, QTL5 and QTL3. For the inner pericarp, fromleft to right the QTLs correspond to QTL1, QTL2, QTL4, QTL5 and QTL3.Marker positions and the distance between them (in centimorgans) areshown below the QTL positions on the X-axis. LOD score is shown on theY-axis.

FIG. 5. Mean fruit firmness determinations on the outer and innerpericarp of selected heterozygous recombinant individuals and parentlines in 2007 and 2008. The Y axis shows maximum load (N) and the X-axisrepresents the line number. (Panel A) outer pericarp 2007; (Panel B)inner pericarp 2007; (Panel C) outer pericarp 2008; (Panel D) innerpericarp 2008.

FIG. 6. Pectinmethylesterase/pectin methylesterase inhibitor (PME)showing high levels of expression in IL2-3 compared with the M82 parent.Normalised mRNA expression levels of PME are shown on the Y axis. Numberof days post anthesis are shown on the X-axis. Key: white bars=IL2-3;solid bars=M82.

FIG. 7 Normalised relative mRNA concentrations of ethylene responsefactor 12 in lines M82, IL2-4, IL2-3, 1088, 301, 769 and 910 at themature green stage (denoted M) or at the breaker stage (denoted B) asmeasured in (Panel A) outer pericarp and (Panel B) inner pericarp.Texture data from the same lines is shown for (Panel C) outer pericarpand (Panel D) inner pericarp. The letter H denotes the line has aheterozygous introgression.

FIG. 8 Normalised relative mRNA concentrations of Pectin methylesterasein lines M82, IL2-3, 124, 142, 301 and 331 at the mature green stage(denoted M) or at the breaker stage (denoted B) as measured in (Panel A)outer pericarp and (Panel B) inner pericarp. Texture data from the samelines is shown for (Panel C) outer pericarp and (Panel D) innerpericarp. The letter H denotes the line has a heterozygousintrogression.

FIG. 9 Normalised relative mRNA concentrations of Dof zinc fingerprotein 6 in lines M82, IL2-4 and IL2-3 at the mature green stage(denoted M) as measured in (Panel A) outer pericarp and (Panel B) innerpericarp.

FIG. 10 (Panel A) Detailed QTL map for outer pericarp showing markerpositions (Panel B) Detailed QTL map for inner pericarp showing markerpositions.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The technical terms and expressions used within the scope of thisapplication are generally to be given the meaning commonly applied tothem in the pertinent art of plant breeding and cultivation if nototherwise indicated herein below.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a plant”includes one or more plants, and reference to “a cell” includes mixturesof cells, tissues, and the like.

As used herein, the term “about” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

An “allele” is understood within the scope of the invention to refer toalternative or variant forms of various genetic units identical orlinked to different forms of a gene or of any kind of identifiablegenetic element, which are alternative in inheritance because they aresituated at the same locus in homologous chromosomes. Such alternativeor variant forms may be the result of single nucleotide polymorphisms,insertions, inversions, translocations or deletions, or the consequenceof gene regulation caused by, for example, chemical or structuralmodification, transcription regulation or post-translationalmodification/regulation. In a diploid cell or organism, the two allelesof a given gene or genetic element typically occupy corresponding locion a pair of homologous chromosomes.

An allele linked to a quantitative trait may comprise alternative orvariant forms of various genetic units including those that areidentical or linked to a single gene or multiple genes or their productsor even a gene disrupting or controlled by a genetic factor contributingto the phenotype represented by said QTL.

As used herein, the term “backcross”, and grammatical variants thereof,refers to a process in which a breeder crosses a hybrid progeny back toone of the parents, for example, a first generation hybrid F1 with oneof the parental genotypes of the F1 hybrid. In some embodiments, abackcross is performed repeatedly, with a progeny individual of onebackcross being itself backcrossed to the same parental genotype.

As used herein, the term “breeding”, and grammatical variants thereof,refer to any process that generates a progeny individual. Breedings canbe sexual or asexual, or any combination thereof. Exemplary non-limitingtypes of breedings include crossings, selfings, doubled haploidderivative generation, and combinations thereof.

For the purpose of the present invention, the term “co-segregation”refers to the fact that the allele for the trait and the allele(s) forthe markers) tend to be transmitted together because they are physicallyclose together on the same chromosome (reduced recombination betweenthem because of their physical proximity) resulting in a non-randomassociation of their alleles. “Co-segregation” also refers to thepresence of two or more traits within a single plant of which at leastone is known to be genetic and which cannot be readily explained bychance.

A “cultivated tomato plant” is understood within the scope of theinvention to refer to a plant that is no longer in the natural state buthas been developed by human care and for human use and/or consumption.“Cultivated tomato plants” are further understood to exclude thosewild-type species which comprise the trait being subject of thisinvention as a natural trait and/or part of their natural genetics.Cultivated tomato plants also typically display resistance to Tobaccomosaic virus, whereas wild-type species do not.

There are 2 major types of tomato growth: determinate and indeterminate.Determinate growth produces “bush” tomatoes and which are bred forcompactness. The entire plant stops growing once the terminal fruitripens, the remainder of the fruit all ripen simultaneously, and thenthe plant dies. Indeterminate growth produces tomatoes that can grow upto 10 feet in height (so-called “vining” tomatoes) and will only stopgrowing when killed by frost. Their fruit ripen rotationally. In atypical plant, all growth arises from the reiteration of modularsympodial units that each produce three leaves and a multifloweredinflorescence. All field-grown varieties of tomato, including M82, aredeterminate plants whose shoots produce an average of six sympodialunits, each harboring a single inflorescence, within which leaf numbergradually decreases before a precocious termination of growth. Ingeneral, determinate tomatoes are suitable for open field production.Semideterminate and indeterminate “cultivated” varieties are suitablefor staked cultivation in the open field or protected nets and forglasshouse cultivation. For the purposes of the present invention, theS. pennellii introgression lines e.g. IL2-3 and IL2-4 are not regardedas cultivated lines, whereas parental line M82 and recombinant linese.g. QTL-NIL 301 are regarded as cultivated lines.

As used herein, the term “dihaploid line”, refers to stable inbred linesissued from anther culture. Some pollen grains (haploid) cultivated onspecific medium and circumstances can develop plantlets containing nchromosomes. These plantlets are then “doubled” and contain 2nchromosomes. The progeny of these plantlets are named “dihaploid” andare essentially not segregating anymore (stable).

As used herein, the term “gene” refers to a hereditary unit including asequence of DNA that occupies a specific location on a chromosome andthat contains the genetic instruction for a particular characteristic ortrait in an organism.

As used herein, the term “genetic architecture at the QTL” refers to agenomic region which is statistically correlated to the phenotypic traitof interest and represents the underlying genetic basis of thephenotypic trait of interest.

“Genetic engineering”, “transformation” and “genetic modification” areall used herein as synonyms for the transfer of isolated and clonedgenes into the DNA, usually the chromosomal DNA or genome, of anotherorganism.

As used herein, the phrases “genetic marker”, “DNA marker” or “molecularmarker” are interchangeable and refer to a feature of an individual'sgenome (e.g. a nucleotide or a polynucleotide sequence that is presentin an individual's genome) that is linked to one or more loci ofinterest. In some embodiments, a genetic marker is polymorphic in apopulation of interest, or the locus occupied by the polymorphism,depending on context. Genetic markers include, for example, singlenucleotide polymorphisms (SNPs), indels (i.e., insertions/deletions),simple sequence repeats (SSRs) restriction fragment length polymorphisms(RFLPs), random amplified polymorphic DNAs (RAPDs), cleaved amplifiedpolymorphic sequence (CAPS) markers, Diversity Arrays Technology (DArT)markers, and amplified fragment length polymorphisms (AFLPs) among manyother examples. Genetic markers can, for example, be used to locategenetic loci containing alleles on a chromosome that contribute tovariability of phenotypic traits. The phrase “genetic marker” can alsorefer to a polynucleotide sequence complementary to a genomic sequence,such as a sequence of a nucleic acid used as probes. A genetic ormolecular marker can be physically located in a position on a chromosomethat is distal or proximal to the genetic loci with which it is linked(i.e. is intragenic or extragenic, respectively). Stated another way,whereas genetic markers are typically employed when the location on achromosome of the gene or of a functional mutation, e.g. within acontrol element outside of a gene, that corresponds to the locus ofinterest has not been identified and there is a very low rate ofrecombination between the genetic marker and the locus of interest, thepresently disclosed subject matter can also employ genetic markers thatare physically within the boundaries of a genetic locus (e.g. inside agenomic sequence that corresponds to a gene such as, but not limited toa polymorphism within an intron or an exon of a gene). In someembodiments of the presently disclosed subject matter, the one or moregenetic markers comprise between one and ten markers, and in someembodiments the one or more genetic markers comprise more than tengenetic markers.

As used herein, the term “genotype” refers to the genetic constitutionof a cell or organism. An individual's “genotype for a set of geneticmarkers” includes the specific alleles, for one or more genetic markerloci, present in the individual's haplotype. As is known in the art, agenotype can relate to a single locus or to multiple loci, whether theloci are related or unrelated and/or are linked or unlinked. In someembodiments, an individual's genotype relates to one or more genes thatare related in that the one or more of the genes are involved in theexpression of a phenotype of interest (e.g. a quantitative trait asdefined herein). Thus, in some embodiments a genotype comprises asummary of one or more alleles present within an individual at one ormore genetic loci of a quantitative trait. In some embodiments, agenotype is expressed in terms of a haplotype (defined herein below).

“Heterozygous” is understood within the scope of the invention to referto unlike alleles at one or more corresponding loci on homologouschromosomes.

“Homozygous” is understood within the scope of the invention to refer tolike alleles at one or more corresponding loci on homologouschromosomes.

As used herein, the term “hybrid” in the context of plant breedingrefers to a plant that is the offspring of genetically dissimilarparents produced by crossing plants of different lines or breeds orspecies, including but not limited to the cross between two inbredlines.

The term “hybridize” as used herein refers to conventional hybridizationconditions, preferably to hybridization conditions at which 5×SSPE, 1%SDS, 1×Denhardts solution is used as a solution and/or hybridizationtemperatures are between 35° C. and 70° C., preferably 65° C. Afterhybridization, washing is preferably carried out first with 2×SSC, 1%SDS and subsequently with 0.2×SSC at temperatures between 35° C. and 75°C. particularly between 45° C. and 65° C., but especially at 59° C.(regarding the definition of SSPE, SSC and Denhardts solution seeSambrook et al. (2001)). High stringency hybridization conditions as forinstance described in Sambrook et al. (2001), are particularlypreferred. Particularly preferred stringent hybridization conditions arefor instance present if hybridization and washing occur at 65° C. asindicated above. Non-stringent hybridization conditions for instancewith hybridization and washing carried out at 45° C. are less preferredand at 35° C. even less.

As used herein, the phrase “inbred line” refers to a geneticallyhomozygous or nearly homozygous population. An inbred line, for example,can be derived through several cycles of brother/sister breedings or ofselfing or in dihaploid production. In some embodiments, inbred linesbreed true for one or more phenotypic traits of interest. An “inbred”,“inbred individual”, or “inbred progeny” is an individual sampled froman inbred line.

As used herein, the terms “introgression”, “introgressed” and“introgressing” refer to the process whereby genes, a QTL or haplotypeof one species, variety or cultivar are moved into the genome of anotherspecies, variety or cultivar, by crossing those species. The crossingmay be natural or artificial. The process may optionally be completed bybackcrossing to the recurrent parent, in which case introgression refersto infiltration of the genes of one species into the gene pool ofanother through repeated backcrossing of an interspecific hybrid withone of its parents. An introgression may also be described as aheterologous genetic material stably integrated in the genome of arecipient plant.

As used herein, the term “linkage”, and grammatical variants thereof,refers to the tendency of alleles at different loci on the samechromosome to segregate together more often than would be expected bychance if their transmission were independent, in some embodiments as aconsequence of their physical proximity. Linkage is measured by percentrecombination between loci (centimorgan, cM).

As used herein, the phrase “linkage group” refers to all of the genes orgenetic traits that are located on the same chromosome. Within thelinkage group, those loci that are close enough together can exhibitlinkage in genetic crosses. Since the probability of crossover increaseswith the physical distance between loci on a chromosome, loci for whichthe locations are far removed from each other within a linkage groupmight not exhibit any detectable linkage in direct genetic tests. Theterm “linkage group” is mostly used to refer to genetic loci thatexhibit linked behavior in genetic systems where chromosomal assignmentshave not yet been made. Thus, in the present context, the term “linkagegroup” is synonymous with the physical entity of a chromosome, althoughone of ordinary skill in the art will understand that a linkage groupcan also be defined as corresponding to a region of (i.e. less than theentirety) of a given chromosome.

“Locus” is understood within the scope of the invention to refer to aregion on a chromosome, which comprises a gene or any other geneticelement or factor contributing to a trait.

As used herein, the term “marker allele” refers to an alternative orvariant form of a genetic unit as defined herein above, when used as amarker to locate genetic loci containing alleles on a chromosome thatcontribute to variability of phenotypic traits.

“Marker-based selection” is understood within the scope of the inventionto refer to e.g. the use of genetic markers to detect one or morenucleic acids from the plant, where the nucleic acid is linked to adesired trait to identify plants that carry genes, QTLs or haplotypesfor desirable (or undesirable) traits, so that those plants can be used(or avoided) in a selective breeding program.

“Marker assisted selection” refers to the process of selecting a desiredtrait or desired traits in a cultivated plant or cultivated plants bydetecting one or more nucleic acids from the cultivated plant, where thenucleic acid is linked to the desired trait.

As used herein, “marker locus” refers to a region on a chromosome, whichcomprises a nucleotide or a polynucleotide sequence that is present inan individual's genome and that is linked to one or more loci ofinterest, which may which comprise a gene or any other genetic elementor factor contributing to a trait. “Marker locus” also refers to aregion on a chromosome, which comprises a polynucleotide sequencecomplementary to a genomic sequence, such as a sequence of a nucleicacid used as a probe.

“Microsatellite or SSRs (Simple sequence repeats) marker” is understoodwithin the scope of the invention to refer to a type of genetic markerthat consists of numerous repeats of short sequences of DNA bases, whichare found at loci throughout the plant's genome and have a likelihood ofbeing highly polymorphic.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammaticalequivalents thereof used herein means at least two nucleotidescovalently linked together. Oligonucleotides are typically from about 7,8, 9, 10, 12, 15, 1820 25, 30, 40, 50 or up to about 100 nucleotides inlength. Nucleic acids and polynucleotides are polymers of any length,including longer lengths, e.g. 200, 300, 500, 1000, 2000, 3000, 5000,7000, 10000, etc. A nucleic acid of the present invention will generallycontain phosphodiester bonds, although in some cases, nucleic acidanalogs are included that may have alternate backbones comprising, e.g.phosphoramidate, phosphorothioate, phosphorodithioate, orO-methylphosphoroamidite linkages (see Eckstein, 1991), and peptidenucleic acid backbones and linkages. Mixtures of naturally occurringnucleic acids and analogs can be used. Particularly preferred analogsfor oligonucleotides are peptide nucleic acids (PNA).

As used herein, the term “offspring” plant refers to any plant resultingas progeny from a vegetative or sexual reproduction from one or moreparent plants or descendants thereof. For instance an offspring plantcan be obtained by cloning or selfing of a parent plant or by crossingtwo parent plants and include selfings as well as the F1 or F2 or stillfurther generations. An F1 is a first-generation offspring produced fromparents at least one of which is used for the first time as donor of atrait, while offspring of second generation (F2) or subsequentgenerations (F3, F4, and the like) are specimens produced from selfingsof F1s, F2s and the like. An F1 can thus be (and in some embodiments is)a hybrid resulting from a cross between two true breeding parents(true-breeding is homozygous for a trait), while an F2 can be (and insome embodiments is) an offspring resulting from self-pollination of theF1 hybrids.

“PCR (Polymerase chain reaction)” is understood within the scope of theinvention to refer to a method of producing relatively large amounts ofspecific regions of DNA or subset(s) of the genome, thereby makingpossible various analyses that are based on those regions.

“PCR primer” is understood within the scope of the invention to refer torelatively short fragments of single-stranded DNA used in the PCRamplification of specific regions of DNA.

As used herein, the expression “phenotype” or “phenotypic trait” refersto the appearance or other detectable characteristic of an individual,resulting from the interaction of its genome, proteome and/or metabolomewith the environment.

A “plant” is any plant at any stage of development, particularly a seedplant.

A “plant cell” is a structural and physiological unit of a plant,comprising a protoplast and a cell wall. The plant cell may be in formof an isolated single cell or a cultured cell, or as a part of higherorganized unit such as, for example, plant tissue, a plant organ, or awhole plant.

“Plant cell culture” means cultures of plant units such as, for example,protoplasts, cell culture cells, cells in plant tissues, pollen, pollentubes, ovules, embryo sacs, zygotes and embryos at various stages ofdevelopment.

“Plant material” refers to leaves, stems, roots, flowers or flowerparts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell ortissue cultures, or any other part or product of a plant.

As used herein, the phrase “plant part” refers to a part of a plant,including single cells and cell tissues such as plant cells that areintact in plants, cell clumps, and tissue cultures from which plants canbe regenerated. Examples of plant parts include, but are not limited to,single cells and tissues from pollen, ovules, leaves, embryos, roots,root tips, anthers, flowers, fruits, stems, shoots, and seeds; as wellas scions, rootstocks, protoplasts, calii, and the like.

“Plant tissue” as used herein means a group of plant cells organizedinto a structural and functional unit. Any tissue of a plant in plantaor in culture is included. This term includes, but is not limited to,whole plants, plant organs, plant seeds, tissue culture and any groupsof plant cells organized into structural and/or functional units. Theuse of this term in conjunction with, or in the absence of, any specifictype of plant tissue as listed above or otherwise embraced by thisdefinition is not intended to be exclusive of any other type of planttissue.

“Polymorphism” is understood within the scope of the invention to referto the presence in a population of two or more different forms of agene, genetic marker, or inherited trait or a gene product obtainable,for example, through alternative splicing, DNA methylation, etc.

As used herein, the term “population” means a genetically heterogeneouscollection of plants sharing a common genetic derivation.

“Probe” as used herein refers to a group of atoms or molecules which iscapable of recognizing and binding to a specific target molecule orcellular structure and thus allowing detection of the target molecule orstructure. Particularly, “probe” refers to a labeled DNA or RNA sequencewhich can be used to detect the presence of and to quantitate acomplementary sequence by molecular hybridization.

As used herein, the term “progeny” refers to the descendant(s) of aparticular cross. Typically, progeny result from breeding of twoindividuals, although some species (particularly some plants andhermaphroditic animals) can be selfed (i.e. the same plant acts as thedonor of both male and female gametes). The descendant(s) can be, forexample, of the F1, the F2, or any subsequent generation.

The term “QTL” is used herein in its art-recognised meaning. The term“QTL linked to increased fruit firmness in tomato” as well as theshorter term “QTL for increased fruit firmness” refer to a regionlocated on a particular chromosome of tomato that is linked to at leastone gene that is responsible for increased fruit firmness or at least aregulatory region, i.e. a region of a chromosome that controls theexpression of one or more genes involved in increased fruit firmness. AQTL may for instance comprise one or more genes, the products of whichconfer increased fruit firmness. Alternatively, a QTL may for instancecomprise regulatory genes or sequences, the products of which influencethe expression of genes on other loci in the genome of the plant therebyconferring the increased fruit firmness. The QTLs of the presentinvention may be defined by indicating their genetic location in thegenome of the respective wild tomato accession using one or moremolecular genomic markers. One or more markers, in turn, indicate aspecific locus. Distances between loci are usually measured by frequencyof crossing-over between loci on the same chromosome. The farther aparttwo loci are, the more likely that a crossover will occur between them.Conversely, if two loci are close together, a crossover is less likelyto occur between them. As a rule, one centimorgan (cM) is equal to 1%recombination between loci (markers). When a QTL can be indicated bymultiple markers the genetic distance between the end-point markers isindicative of the size of the QTL.

As used herein, the terms “QTL1”, “QTL2”, “QTL3”, “QTL4” and “QTL5”refer to the genomic regions linked to increased tomato firmness asdefined by the markers NT3853, NT3907 and TG14; HOX7A and CT277; HB2600,and TG353; Lm0127 and Lm1650; and LE5100 and LE5200 respectively. Forthe purposes of the instant disclosure, these markers are said to bepresent on S. pennellii chromosome 2.

The term “quantitatively determining” is defined herein as establishingor assessing in a manner involving measurement, in particular themeasurement of aspects measurable in terms of amounts and number.Determinations in degrees of severity and indications of greater, more,less, or equal or of increasing or decreasing magnitude, are notcomprised in the present term “quantitatively determining”, which termultimately implies the presence of objective counting mechanism fordetermining absolute values.

The term “recipient tomato plant” is used herein to indicate a tomatoplant that is to receive DNA obtained from a donor tomato plant thatcomprises a QTL for increased fruit firmness. Said “recipient tomatoplant” may or may not already comprise one or more QTLs for fruitfirmness, in which case the term indicates a plant that is to receive anadditional QTL.

The term “natural genetic background” is used herein to indicate theoriginal genetic background of a QTL. Such a background may for instancebe the genome of a wild accession of tomato. For instance, the QTLs ofthe present invention were found at specific locations on chromosome 2of S. pennellii. As an example, S. pennellii represents the naturalgenetic background of QTLs 1, 2, 3, 4 and 5 on chromosome 2 of S.pennellii. Conversely, a method that involves the transfer of DNAcomprising these QTLs, or a fruit firmness-conferring part thereof, fromChromosome 2 of S. pennellii to the same position on chromosome 2 ofanother tomato species, preferably cultivated S. lycopersicum, willresult in these QTLs, or said fruit firmness-conferring part thereof,not being in its natural genetic background.

In this application, a “recombination event” is understood to mean ameiotic crossing-over.

“Sequence Homology” or “sequence Identity” is used hereininterchangeably. The terms “identical” or “percent identity” in thecontext of two or more nucleic acid or protein sequences, refer to twoor more sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection,if two sequences which are to be compared with each other differ inlength, sequence identity preferably relates to the percentage of thenucleotide residues of the shorter sequence which are identical with thenucleotide residues of the longer sequence. Sequence identity can bedetermined conventionally with the use of computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive Madison, Wis. 53711). Bestfit utilizes the local homologyalgorithm of Smith and Waterman (1981) in order to find the segmenthaving the highest sequence identity between two sequences. When usingBestfit or another sequence alignment program to determine whether aparticular sequence has for instance 95% identity with a referencesequence of the present invention, the parameters are preferably soadjusted that the percentage of identity is calculated over the entirelength of the reference sequence and that homology gaps of up to 5% ofthe total number of the nucleotides in the reference sequence arepermitted. When using Bestfit, the so called optional parameters arepreferably left at their preset (“default”) values. The deviationsappearing in the comparison between a given sequence and the abovedescribed sequences of the invention may be caused for instance byaddition, deletion, substitution, insertion or recombination. Such asequence comparison can preferably also be carried out with the programfasta20u66” (version 2.0u65, September 1998 by William R. Pearson andthe University of Virginia; see also W. R. Pearson (1990), appendedexamples and at workbench.sdsc.edu/). For this purpose, the “default”parameter settings may be used.

Another indication that two nucleic acid sequences are substantiallyidentical is that the two molecules hybridize to each other understringent conditions. The phrase “hybridizing specifically to” refers tothe binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g. total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

As used herein, the phrases “sexually crossed” and “sexual reproduction”in the context of the presently disclosed subject matter refers to thefusion of gametes to produce progeny (e.g. by fertilization, such as toproduce seed by pollination in plants). A “sexual cross” or“cross-fertilization” is in some embodiments fertilization of oneindividual by another (e.g. cross-pollination in plants). The term“selfing” refers in some embodiments to the production of seed byself-fertilization or self-pollination i.e. pollen and ovule are fromthe same plant.

A “single nucleotide polymorphism” (SNP) is a DNA sequence variationoccurring when a single nucleotide A, C, G, T in the genome (or othershared sequences as mitochondrial DNA) differs between a set (paired)chromosomes of an individual or differs between members of a species.

The term “standard greenhouse conditions” and “standard conditions”refer to the conditions of light, humidity, temperature, etc whereuponplants are grown or incubated, for instance for the purpose ofphenotypic characterization of disease fruit firmness, as beingstandard. Growth conditions can for example be a photoperiod of 16 h(photosynthetic photon flux (PPF) 50 to 1000 μmol nv2 s1), preferably aregime of 8 hours dark, an air temperature of about 20° C. during theday and 18° C. at night, a water vapour pressure deficit of about 4.4 gm3 corresponding to a relative humidity (RH) of about 60%-85%, atatmospheric oxygen concentration and at atmospheric air pressure(generally 1008 hPa). Water and nutrients may be given drop wise nearthe stem, or in the form of spray or mist.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology-Hybridization withNucleic Acid Probes part 1 chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays” Elsevier,New York. Generally, highly stringent hybridization and wash conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength and pH. Typically,under “stringent conditions” a probe will hybridize to its targetsubsequence, but to no other sequences.

The Tm is the temperature (under defined ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe. Verystringent conditions are selected to be equal to the Tm for a particularprobe. An example of stringent hybridization conditions forhybridization of complementary nucleic acids which have more than 100complementary residues on a filter in a Southern or Northern blot is 50%formamide with 1 mg of heparin at 42° C., with the hybridization beingcarried out overnight. An example of highly stringent wash conditions is0.15M NaCl at 72° C. for about 15 minutes. An example of stringent washconditions is a 0.2 times SSC wash at 65° C. for 15 minutes (seeSambrook, infra, for a description of SSC buffer). Often, a highstringency wash is preceded by a low stringency wash to removebackground probe signal. An example medium stringency wash for a duplexof for example more than 100 nucleotides, is 1 times SSC at 45° C. for15 minutes. An example low stringency wash for a duplex of, e.g. morethan 100 nucleotides, is 4-6 times SSC at 40° C. for 15 minutes. Forshort probes (e.g. about 10 to 50 nucleotides), stringent conditionstypically involve salt concentrations of less than about 1.0M Na ionconcentration, typically about 0.01 to 1.0 M Na ion concentration (orother salts) at pH 7.0 to 8.3, and the temperature is typically at leastabout 30° C. Stringent conditions can also be achieved with the additionof destabilizing agents such as formamide. In general, a signal to noiseratio of 2 times (or higher) than that observed for an unrelated probein the particular hybridization assay indicates detection of a specifichybridization. Nucleic acids that do not hybridize to each other understringent conditions are still substantially identical if the proteinsthat they encode are substantially identical. This occurs for examplewhen a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

As used herein, the term “tomato” means any plant, line or populationwithin the species Solanum lycopersicum (synonyms are Lycopersiconlycopersicum or Lycopersicon esculentum) or formerly known under thegenus name of Lycopersicon including but not limited to L. cerasiforrne,L. cheesmanii, L. chilense, L. chmielewskii, L. esculentum (now S.pennellii), L. hirsutum, L. parviborum, L. pennellii, L. peruvianum, L.pimpinellifolium, or S. lycopersicoides. The newly proposed scientificname for L. esculentum is S. pennellii. Similarly, the names of the wildspecies may be altered. L. pennellii has become S. pennellii, L.hirsutum may become S. habrochaites, L. peruvianum may be split into S.‘N peruvianum’ and S. ‘Callejon de Hueyles’, S. peruvianum, and S.corneliomuelleri, L. parviflorum may become S. neorickii, L.chmielewskii may become S. chmielewskii, L. chilense may become S.chilense, L. cheesmaniae may become S. cheesmaniae or S. galapagense,and L. pimpinellifolium may become S. pimpinellifolium (Knapp (2005)).

“Trait” is understood within the scope of the invention to refer to acharacteristic or phenotype, for example increased fruit firmness. Atrait may be inherited in a dominant or recessive manner, or may bemonogenic or polygenic.

“Monogenic” is understood within the scope of the invention to refer tobeing determined by a single locus.

“Polygenic” is understood within the scope of the invention to refer tobeing determined by more than one locus.

“Dominant” is understood within the scope of the invention to refer toan allele which determines the phenotype when present in theheterozygous or homozygous state.

A “recessive” allele is only displayed when present in the homozygousstate.

“Isogenic” is understood within the scope of the invention to refer tocultivated plants which are genetically identical, except that they maydiffer by the presence or absence of a heterologous DNA sequence.

“Harvesting stage” is understood within the scope of the invention tomean the date of harvesting ie the date the tomato fruit is removed fromthe plant.

“Immature Green stage” is defined as when the fruits are unripe andstill growing in size. This stage is understood to be the first stage inthe ripening process.

“Mature green stage” is defined as when the fruit is fully expandedmature, but unripe and follows the “immature green stage” in theripening process. Mature green tomatoes have a white to yellow “star” onthe blossom end. Traditional tomatoes harvested at the mature greenstage are best suited for the commercial fresh market because theytolerate rough handling better than the riper stages and hold theirshape the longest in storage, shipping, and on the supermarket shelf;however they somehow lack full aroma and taste.

“Breaker stage” is defined as first sign of red colour in the fruit,typically it occurs within 24 hours of the mature green stage. Tomatoesthat are harvested at the “Breaker stage” usually have better flavor andtaste but they have reduced firmess and are slightly less suitable forhandling, packaging and transportation than tomatoes at the mature greenstage.

“Red ripe stage” is defined as when the fruits are fully red, with nosign of green colour. These fruits have reached their optimum in tasteand flavor but they cannot be transported because of their lack offirmness and they do not tolerate much handling.

“Genetic element” and “genetic element, or part thereof” are understoodwithin the scope of the invention to mean a QTL or part thereof (inparticular, a gene residing on the chromosome under the QTL) that iscapable of contributing to the firmness of the fruits of the plant byinfluencing expression of the firmness trait at the level of the DNAitself, at the level of translation, transcription and/or activation ofa final polypeptide product, i.e., to regulate metabolism in tomatofruit flesh leading to the phenotypic expression of the genotype.

“Inner pericarp” and “outer pericarp” are understood within the scope ofthe invention to mean fruit tissue where the outer pericarp is the layer(approximately 2 mm) immediately below the outer epidermis and above thevascular tissue layer. The inner pericarp is from 3 mm up to 10 mm belowthe vascular layer and before the inner epidermis.

“Commercially desirable characteristics” are understood within the scopeof the invention to include but not be limited to superior fruitquality, disease resistance, insect resistance, uniform shape and size

“Harvesting slot” is understood within the scope of the invention tomean the period of time from the harvesting stage until when the fruitis too ripe to be harvested for the purposes of commercial sale.Typically, the harvesting slot starts at mature green stage andcontinues until the breaker stage plus two to five days, depending onthe cultivar and environmental conditions.

“donor tomato plant” is understood within the scope of the invention tomean the tomato plant which provides at least one genetic element linkedto significantly increased fruit firmness.

“linked to” and “characterized by” or “associated with” at least one ofthe DNA markers of the present invention is understood within the scopeof the invention to mean a DNA sequence that is genetically linked, tothe genetic element responsible for the increased fruit firmness traitand wherein a specific marker sequence is linked to a particular alleleof that gene. When two markers/sequences are said to be geneticallylinked, the recombination frequency between the two markers/sequencesare low and it can be expected that both these markers/sequences areinherited jointly. For the population of plants described herein,markers named as linked to the QTLs are a distance of 1 cM or less away.Markers that are 1 cM distance apart from each other have a 1% chance ofbeing separated from each other due to a recombination event in a singlegeneration.

“fruit firmness conferring parts of QTLs” is understood within the scopeof the invention to mean the genetic element(s) or part(s) thereofresponsible for increased tomato fruit firmness as determined bymechanical measurement as described in the examples.

“Increase in fruit firmness” and “increased fruit firmness” areunderstood within the scope of the invention to mean tomato fruit whichhas an increased maximum load value (for example as described in Example1), statistically significant at P<0.05 or P<0.01 compared to fruit froma control plant.

Maximum load is defined as the value that represents the greatest load(in Newtons (N)) required to cause failure of tissue integrity.

“control tomato plant” is understood within the scope of the inventionto mean a tomato plant that has the same genetic background as thecultivated tomato plant of the present invention wherein the controlplant does not have any of the at least one genetic elements—or partthereof—of the present invention linked to increased fruit firmness. Inparticular a control tomato plant is a tomato plant belonging to thesame plant variety and does not comprise any of the at least one geneticelement, or part thereof. The control tomato plant is grown for the samelength of time and under the same conditions as the cultivated tomatoplant of the present invention. Plant variety is herein understoodaccording to definition of UPOV. Thus a control tomato plant may be aninbred line or a hybrid provided that they have the same geneticbackground as the tomato plant of the present invention except thecontrol plant does not have any of the at least one genetic element—orpart thereof—of the present invention linked to increased fruitfirmness.

“anthesis” is understood within the scope of the invention to mean theperiod during which the flower is fully open and pollen is released.

“Processed food” is understood within the scope of the invention to meanfood which has been altered from its natural state. Methods used forprocessing food include but are not limited to canning, freezing,refrigeration, dehydration and asceptic processing.

“Fresh cut market” is understood within the scope of the invention tomean vegetables on the market which have been minimally processed.

“first true leaf” is understood within the scope of the invention tomean when the first leaf emerges after emergence of the seed leaves orcotyledons.

Plant Breeding

The purpose of breeding programs in agriculture and horticulture is toenhance the performance of plants by improving their geneticcomposition. In essence, this improvement accrues by increasing thefrequency of the most favorable alleles for the genes influencing theperformance characteristics of interest.

Wild plant lines provide a rich resource of genetic and phenotypicvariation. Traditionally, agricultural or horticultural practice makesuse of this variation by selecting a wild plant line or its offspringfor having desired genotypic or potential phenotypic properties,crossing it with a line having additional desired genotypic or potentialphenotypic properties and selecting from among the offspring plantsthose that exhibit the desired genotypic or potential phenotypicproperties (or an increased frequency thereof).

A growing understanding and utilization of the laws of Mendelianinheritance in combination with molecular genetic tools have in the pastcentury facilitated this selection process. For example, methods forselecting plants for having desired genotypic or potential phenotypicproperties have become available based on testing the plant for thepresence of a quantitative trait locus (QTL); i.e. for the presence of agenetic element containing alleles linked to the expression of acontinuously distributed (quantitative) phenotypic trait. Usually a QTLis characterized by one or more markers that statistically associate tothe quantitative variation in the phenotypic trait and is essentiallysynonymous to a gene. QTL mapping allows for the identification ofgenetic element(s) affecting the expression of a trait of interest. Inplant breeding, it allows for marker-assisted selection (MAS); i.e. theselection of plants having favorable alleles by detecting in thoseplants the QTL-associated markers.

Knowledge of the inheritance of various traits would allow for theselection of lines homozygous for a QTL linked to increased fruitfirmness. Use of the knowledge of the genetic origin and location of adesired trait in a breeding program can increase the accuracy of thepredicted breeding outcome and can enhance the rate of selectioncompared to conventional breeding programs. For instance, the fact thatthe genetic basis of a desired trait is heritably linked to anothertrait can help to increase uniformity for those two traits among theoffspring since a parent homozygous for the desired alleles will passthem to most if not all offspring, resulting in a reduced segregation inthe offspring.

The presently disclosed subject matter provides for better models formarker-assisted selection (MAS). The presently disclosed subject matterrelates to methods of plant breeding and to methods to select tomatoplants, particularly cultivated tomato plants as breeder plants for usein breeding programs or cultivated tomato plants having desiredgenotypic or potential phenotypic properties, in particular those whichproduce tomato fruit with increased fruit firmness at the harvestingstage.

Accordingly, there is provided a tomato fruit with increased fruitfirmness at the harvesting stage linked to at least one genetic elementin the cultivated tomato plant producing said tomato, wherein saidfirmness is from between 1.2 to 2.0 times greater than that of fruitfrom a control tomato plant which does not have the said at least onegenetic element.

The harvesting stage is preferably the mature green stage.Alternatively, the harvesting stage can be any chosen point in thedevelopment of the tomato fruit, which includes but is not limited tothe immature green stage, rapid expansion stage, mature green stage,breaker stage or red ripe stage or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 daysafter any of these stages. In one embodiment, the harvesting stage isthe breaker stage plus 7 days.

In a specific embodiment there is provided a tomato fruit providingincreased fruit firmness at the mature green stage linked to at leastone genetic element in the cultivated tomato plant producing saidtomato, wherein said firmness is from between 1.2 to 2.0 times greaterthan that of fruit from a control tomato plant which does not have thesaid at least one genetic element. In another embodiment, said firmnessis 1.2 to 1.5 times greater than that of fruit from a control tomatoplant which does not have the said at least one genetic element.

In a specific embodiment, there is provided a tomato fruit providingincreased fruit firmness at the breaker stage plus 7 days caused by atleast one genetic element in the cultivated tomato plant producing saidtomato, wherein said firmness is from between 1.2 to 2.0 times greaterthan that of fruit from a control tomato plant which does not have thesaid at least one genetic element. In another embodiment, said firmnessis 1.2 to 1.5 times greater than that of fruit from a control tomatoplant which does not have the said at least one genetic element.

Fruit firmness is preferably measured in the outer pericarp and innerpericarp of the cultivated tomato plant. Alternatively, fruit firmnesscan be measured in the inner pericarp only. Preferably, fruit firmnessis measured by mechanical means. Typically, a Lloyd LRX machine is usedas described in the Example section. Such measurements represent asensitive mechanical measure of fruit firmness that correlates very wellwith conventional penetrometer assays, but gives substantially moresensitive and reproducible results. In addition these measurements alsocorrelate well with organoleptic/sensory descriptions of fruit firmness(King et al, 2001). However, alternative methods for measuring fruitfirmness which are known to the skilled person may also be employed suchas those described in Causse et al (2002), the full contents of whichare incorporated herein in their entirety. Preferably the control tomatoplant is M82 with deposit number NCIMB 41661.

Alternatively, the control plant can be any tomato plant which differsfrom its offspring essentially due to the absence of at least onegenetic element, or part thereof, responsible for increased fruitfirmness in the cultivated tomato plant. The control tomato plant may beselected from any plant, line or population within the species Solanumlycopersicum (synonyms are Lycopersicon lycopersicum or Lycopersiconesculentum) or formerly known under the genus name of Lycopersiconincluding but not limited to L. cerasiforrne, L. cheesmanii, L.chilense, L. chmielewskii, L. esculentum (now S. pennellii), L.hirsutum, L. parviborum, L. pennellii, L. peruvianum, L.pimpinellifolium, or S. lycopersicoides. The newly proposed scientificname for L. esculentum is S. pennellii. Similarly, the names of the wildspecies may be altered. L. pennellii has become S. pennellii, L.hirsutum may become S. habrochaites, L. peruvianum may be split into S.‘N peruvianum’ and S. ‘Callejon de Hueyles’, S. peruvianum, and S.corneliomuelleri, L. parviflorum may become S. neorickii, L.chmielewskii may become S. chmielewskii, L. chilense may become S.chilense, L. cheesmaniae may become S. cheesmaniae or S. galapagense,and L. pimpinellifolium may become S. pimpinellifolium (Knapp (2005))

Preferably the genetic element(s) responsible for significantlyincreased fruit firmness of the present invention is present onchromosome 2 of tomato. Preferably, the at least one genetic element ispresent on chromosome 2 of S. pennellii. More preferably, the at leastone genetic element is present on the long arm of chromosome 2 of S.pennellii. Most preferably, the at least one genetic element is presenton the long arm of chromosome 2 of S. pennellii and is any one, two,three, four or all of QTLs 1 to 5.

In a further aspect, there is provided a cultivated tomato plant whichproduces tomato fruit as described herein wherein said plant comprisesat least one genetic element which is characterized by at least one DNAmarker selected from the group consisting of NT3853, NT3907, TG14,HOX7A, CT277, HB2600, TG353, Lm0127, Lm1650, LE5100 and LE5200.

In a further aspect, there is provided a cultivated tomato plant whichproduces tomato fruit wherein said plant comprises at least one geneticelement which is complementary to the corresponding genetic element inS. pennellii lines IL2-3 and/or IL2-4 deposited under accession numbersLA4038 and LA4039, respectively, wherein said genetic element in S.pennellii IL2-3 and/or IL2-4 can be characterized by at least one DNAmarker selected from the group consisting of NT3853, NT3907, TG14,HOX7A, CT277, HB2600, TG353, Lm0127, Lm1650, LE5100 and LE5200.

There is also provided a cultivated tomato plant which produces tomatofruit with increased fruit firmness compared to fruit from a controltomato plant wherein said cultivated tomato plant has one geneticelement which is a QTL selected from a) QTL1 linked to at least one ofthe DNA markers NT3853, NT3907 and TG14; or b) QTL2 linked to at leastone of the DNA markers HOX7A and CT277; or c) QTL3 linked to at leastone of the DNA markers HB2600, TG353; or d) QTL4 linked to at least oneof the DNA markers Lm0127 and Lm1650; or e) QTL5 linked to at least oneof the DNA markers LE5100 and LE5200.

There is also provided a cultivated tomato plant which produces tomatofruit with increased fruit firmness compared to fruit from a controltomato plant wherein said cultivated tomato plant comprises at least onegenetic element, wherein the genetic elements are QTL1 linked to atleast one of the DNA markers NT3853, NT3907 and TG14; and QTL2 linked toat least one of the DNA markers HOX7A and CT277.

There is also provided a cultivated tomato plant produces tomato fruitwith increased fruit firmness compared to a control tomato plant whereinsaid cultivated tomato plant comprises at least one genetic element,wherein the genetic elements are QTL1 linked to at least one of the DNAmarkers NT3853, NT3907 and TG14; and QTL3 linked to at least one of theDNA markers HB2600, and TG353.

There is also provided a cultivated tomato plant produces tomato fruitwith increased fruit firmness compared to a control tomato plant whereinsaid cultivated tomato plant comprises at least one genetic element,wherein the genetic elements are QTL1 linked to at least one of the DNAmarkers NT3853, NT3907 and TG14; and QTL4 linked to at least one of theDNA markers Lm0127 and Lm1650.

There is also provided a cultivated tomato plant produces tomato fruitwith increased fruit firmness compared to a control tomato plant whereinsaid cultivated tomato plant comprises at least one genetic element,wherein the genetic elements are QTL1 linked to at least one of the DNAmarkers NT3853, NT3907 and TG14; and QTL5 linked to at least one of theDNA markers LE5100 and LE5200

There is also provided a cultivated tomato plant produces tomato fruitwith increased fruit firmness compared to a control tomato plant whereinsaid cultivated tomato plant comprises at least one genetic element,wherein the genetic elements are QTL2 linked to at least one of the DNAmarkers HOX7A and CT277; and QTL3 linked to at least one of the DNAmarkers HB2600, and TG353.

There is also provided a cultivated tomato plant produces tomato fruitwith increased fruit firmness compared to a control tomato plant whereinsaid cultivated tomato plant comprises at least one genetic element,wherein the genetic elements are QTL2 linked to at least one of the DNAmarkers HOX7A and CT277; and QTL4 linked to at least one of the DNAmarkers Lm0127 and Lm1650.

There is also provided a cultivated tomato plant produces tomato fruitwith increased fruit firmness compared to a control tomato plant whereinsaid cultivated tomato plant comprises at least one genetic element,wherein the genetic elements are QTL2 linked to at least one of the DNAmarkers HOX7A and CT277; and QTL5 linked to at least one of the DNAmarkers LE5100 and LE5200.

There is also provided a cultivated tomato plant produces tomato fruitwith increased fruit firmness compared to a control tomato plant whereinsaid cultivated tomato plant comprises at least one genetic element,wherein the genetic elements are QTL3 linked to at least one of the DNAmarkers HB2600 and TG353; and QTL4 linked to at least one of the DNAmarkers Lm0127 and Lm1650.

There is also provided a cultivated tomato plant produces tomato fruitwith increased fruit firmness compared to a control tomato plant whereinsaid cultivated tomato plant comprises at least one genetic element,wherein the genetic elements are QTL3 linked to at least one of the DNAmarkers HB2600 and TG353; and QTL5 linked to at least one of the DNAmarkers LE5100 and LE5200.

There is also provided a cultivated tomato plant produces tomato fruitwith increased fruit firmness compared to a control tomato plant whereinsaid cultivated tomato plant comprises at least one genetic element,wherein the genetic elements are QTL4 linked to at least one of the DNAmarkers Lm0127 and Lm1650; and QTL5 linked to at least one of the DNAmarkers LE5100 and LE5200.

There is also provided a cultivated tomato plant produces tomato fruitwith increased fruit firmness compared to a control tomato plant whereinsaid cultivated tomato plant comprises at least one genetic element,wherein the genetic elements are QTL1 linked to at least one of the DNAmarkers NT3853, NT3907 and TG14; and QTL2 linked to at least one of theDNA markers HOX7A and CT277; and QTL3 linked to at least one of the DNAmarkers HB2600, TG353; and QTL4 linked to at least one of the DNAmarkers Lm0127 and Lm1650; and QTL5 linked to at least one of the DNAmarkers LE5100 and LE5200.

There is also provided a cultivated tomato plant produces tomato fruitwith increased fruit firmness compared to a control tomato plant whereinsaid cultivated tomato plant comprises at least one genetic element,wherein the genetic element is QTL1 linked to at least one of the DNAmarkers NT3853, NT3907 and TG14; which is present only in the innerpericarp.

In a further aspect of the invention, there is provided a cultivatedtomato plant as herein described wherein said plant is an inbred, adihaploid or a hybrid. In a specific embodiment, the cultivated tomatoplant is male sterile.

In a further aspect of the invention, there is provided tomato seedwhich produces a cultivated tomato plant as herein described.

In a further aspect of the invention, there is provided a plant part ofa cultivated tomato plant as herein described.

In a further aspect, there is provided plant material obtainable from aplant part of a cultivated tomato plant as herein described.

Identification of QTLs Linked to Increased Fruit Firmness in Tomato

The presently disclosed subject matter also provides methods forselecting a cultivated tomato plant with fruit having increased firmnesscompared to fruit from a control tomato plant. Such methods comprisedetecting in the cultivated tomato plant the presence of one or more ofQTLs 1 to 5 as defined herein. In general, the methods compriseproviding a sample of genomic DNA from a tomato plant; and (b) detectingin the sample of genomic DNA at least one molecular marker linked to aQTL

selected from the group consisting of QTLs 1 to 5. In some embodiments,the detection step may comprise detecting at least one molecular markerfrom the group, the at least one molecular markers detecting at leastone of QTLs 1 to 5. The providing of a sample of genomic DNA from atomato plant can be performed by standard DNA isolation methods wellknown in the art.

In some embodiments, the detecting of a molecular marker (step (b)) maycomprise the use of a nucleic acid probe having a base sequence that issubstantially complementary to the nucleic acid sequence defining themolecular marker and which nucleic acid probe specifically hybridizesunder stringent conditions with a nucleic acid sequence defining themolecular marker. A suitable nucleic acid probe can for instance be asingle strand of the amplification product corresponding to the marker.The detecting of a molecular marker can also comprise performing anucleic acid amplification reaction on the genomic DNA to detect one ormore QTLs. This can be done by performing a PCR reaction using a set ofmarker-specific primers. In some embodiments, the detecting can comprisethe use of at least one set of primers defining one or more markerslinked to one or more of QTLS 1 to 5, or a set of primers whichspecifically hybridize under stringent conditions with nucleic acidsequences of one or more markers linked to one or more of QTLS 1 to 5.

The presently disclosed methods can also include detecting an amplifiedDNA fragment linked to the presence of a QTL. In some embodiments, theamplified fragment linked to presence of a QTL has a predicted length ornucleic acid sequence, and detecting an amplified DNA fragment havingthe predicted length or the predicted nucleic acid sequence is performedsuch that the amplified DNA fragment has a length that corresponds (plusor minus a few bases; e.g. a length of one, two or three bases more orless) to the expected length as based on a similar reaction with thesame primers with the DNA from the plant in which the marker was firstdetected or the nucleic acid sequence that corresponds (has a homologyof in some embodiments more than 80%, in some embodiments more than 90%,in some embodiments more than 95%, in some embodiments more than 97%,and in some embodiments more than 99%) to the expected sequence as basedon the sequence of the marker linked to that QTL in the plant in whichthe marker was first detected. One of ordinary skill in the art wouldappreciate that markers that are absent in plants providing fruit withincreased fruit firmness, while they were present in the controlparent(s) (so-called trans-markers), can also be useful in assays fordetecting increased fruit firmness among offspring plants, althoughtesting the absence of a marker to detect the presence of a specifictrait is not optimal. The detecting of an amplified DNA fragment havingthe predicted length or the predicted nucleic acid sequence can beperformed by any of a number of techniques, including but not limited tostandard gel-electrophoresis techniques or by using automated DNAsequencers. These methods are well known to the skilled person.

Molecular Markers and QTLs

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization can be due to DNA-DNAhybridization techniques after digestion with a restriction enzyme(RFLP) and/or due to techniques using the polymerase chain reaction(e.g. STS, SSR/microsatellites, AFLPs and the like). In someembodiments, all differences between two parental genotypes segregate ina mapping population based on the cross of these parental genotypes. Thesegregation of the different markers can be compared and recombinationfrequencies can be calculated. Methods for mapping markers in plants aredisclosed in, for example, Glick & Thompson, 1993; Zietkiewicz et al.,1994.

The recombination frequencies of molecular markers on differentchromosomes are generally 50%. Between molecular markers located on thesame chromosome, the recombination frequency generally depends on thedistance between the markers. A low recombination frequency correspondsto a low genetic distance between markers on a chromosome. Comparing allrecombination frequencies results in the most logical order of themolecular markers on the chromosomes. This most logical order can bedepicted in a linkage map (Paterson, 1996). A group of adjacent orcontiguous markers on the linkage map that is linked to an increasedlevel of fruit firmness can provide the position of a QTL linked toincreased fruit firmness.

The markers identified herein can be used in various aspects of thepresently disclosed subject matter. Aspects of the presently disclosedsubject matter are not to be limited to the use of the markersidentified herein, however. It is stressed that the aspects can alsomake use of markers not explicitly disclosed herein or even yet to beidentified. Other than the genetic unit “gene”, on which the phenotypicexpression depends on a large number of factors that cannot bepredicted, the genetic unit “QTL” denotes a region on the genome that isdirectly related to a phenotypic quantifiable trait.

The five QTLs identified herein are located on chromosome 2 of tomatoand their locations can be characterized by a number of otherwisearbitrary markers. In the present investigations, microsatellite markers(e.g. SSRs) and single nucleotide polymorphisms (SNPs) were used,although restriction fragment length polymorphism (RFLP) markers,amplified fragment length polymorphism (AFLP) markers, insertionmutation markers, sequence-characterized amplified region (SCAR)markers, cleaved amplified polymorphic sequence (CAPS) markers orisozyme markers or combinations of these markers might also have beenused.

In general, a QTL can span a region of several million bases. Therefore,providing the complete sequence information for the QTL is practicallyunfeasible but also unnecessary, as the way in which the QTL is firstdetected—through the observed correlation between the presence of astring of contiguous genomic markers and the presence of a particularphenotypic trait—allows one to trace among a population of offspringplants those plants that have the genetic potential for exhibiting aparticular phenotypic trait. By providing a non-limiting list ofmarkers, the presently disclosed subject matter thus provides for theeffective use of the presently disclosed QTLs in a breeding program.

In general, a marker is specific for a particular line of descent. Thus,a specific trait can be linked to a particular marker. The markers asdisclosed herein not only indicate the location of the QTL, they alsocorrelate with the presence of the specific phenotypic trait in a plant.It is noted that the contiguous genomic markers that indicate thelocation of the QTL on the genome are in principal arbitrary ornon-limiting. In general, the location of a QTL is indicated by acontiguous string of markers that exhibit statistical correlation to thephenotypic trait. Once a marker is found outside that string (i.e. onethat has a LOD-score below a certain threshold, indicating that themarker is so remote that recombination in the region between that markerand the QTL occurs so frequently that the presence of the marker doesnot correlate in a statistically significant manner to the presence ofthe phenotype) the boundaries of the QTL can be considered set. Thus, itis also possible to indicate the location of the QTL by other markerslocated within that specified region. It is further noted that thecontiguous genomic markers can also be used to indicate the presence ofthe QTL (and thus of the phenotype) in an individual plant, whichsometimes means that they can be used in marker-assisted selection (MAS)procedures. In principle, the number of potentially useful markers islimited but can be very large, and one of ordinary skill in the art caneasily identify markers in addition to those specifically disclosed inthe present application. Any marker that is linked to the QTL (e.g.falling within the physical boundaries of the genomic region spanned bythe markers having established LOD scores above a certain thresholdthereby indicating that no or very little recombination between themarker and the QTL occurs in crosses, as well as any marker in linkagedisequilibrium to the QTL, as well as markers that represent the actualcausal mutations within the QTL) can be used in MAS procedures. Thismeans that the markers identified in the application as being linkedwith the QTLs, such as the markers NT3853, NT3907, TG14, HOX7A, CT277,HB2600, TG353, Lm0127, Lm1650, LE5100 and LE5200, are mere examples ofmarkers suitable for use in MAS procedures. Moreover, when the QTL, orthe specific trait-conferring part thereof, is introgressed into anothergenetic background (i.e. into the genome of another tomato or anotherplant species), then some markers might no longer be found in theoffspring although the trait is present therein, indicating that suchmarkers are outside the genomic region that represents the specifictrait-conferring part of the QTL in the original parent line only andthat the new genetic background has a different genomic organization.Such markers of which the absence indicates the successful introductionof the genetic element in the offspring are called “trans markers” (seeabove).

Upon the identification of a QTL, the QTL effect (i.e. for increasedfruit firmness) can for instance be confirmed by assessing fruitfirmness in progeny segregating for the QTLs under investigation. Theassessment of the fruit firmness can suitably be performed by measuringfruit firmness as known in the art.

The markers provided by the presently disclosed subject matter can beused for detecting the presence of one or more increased fruit firmnessalleles at QTLs of the presently disclosed subject matter in a tomatoplant with increased fruit firmness, and can therefore be used inmethods involving marker-assisted breeding and selection of tomatoplants with increased fruit firmness. In some embodiments, detecting thepresence of a QTL of the presently disclosed subject matter is performedwith at least one of the markers for a QTL as defined herein. Thepresently disclosed subject matter therefore relates in another aspectto a method for detecting the presence of a QTL for increased fruitfirmness, comprising detecting the presence of a nucleic acid sequenceof the QTL in a tomato plant, which presence can be detected by the useof the herein disclosed markers.

The nucleotide sequence of a QTL of the presently disclosed subjectmatter can for instance be resolved by determining the nucleotidesequence of one or more markers linked to the QTL and designing internalprimers for the marker sequences that can then be used to furtherdetermine the sequence of the QTL outside of the marker sequences. Forinstance, the nucleotide sequence of the SSR markers disclosed hereincan be obtained by isolating the markers from the electrophoresis gelused in the determination of the presence of the markers in the genomeof a subject plant, and determining the nucleotide sequence of themarkers by, for example, dideoxy chain termination sequencing methods,which are well known in the art. In embodiments of such methods fordetecting the presence of a QTL in a tomato plant, the method can alsocomprise providing a oligonucleotide or polynucleotide capable ofhybridizing under stringent hybridization conditions to a nucleic acidsequence of a marker linked to the QTL, in some embodiments selectedfrom the markers disclosed herein, contacting the oligonucleotide orpolynucleotide with digested genomic nucleic acid of a tomato plant, anddetermining the presence of specific hybridization of theoligonucleotide or polynucleotide to the digested genomic nucleic acid.

In some embodiments, the method is performed on a nucleic acid sampleobtained from the tomato plant, although in situ hybridization methodscan also be employed. Alternatively, one of ordinary skill in the artcan, once the nucleotide sequence of the QTL has been determined, designspecific hybridization probes or oligonucleotides capable of hybridizingunder stringent hybridization conditions to the nucleic acid sequence ofthe QTL and can use such hybridization probes in methods for detectingthe presence of a QTL disclosed herein in a tomato plant.

In a further aspect of the invention, there is provided a method fordetecting a QTL linked to significantly increased fruit firmness infruit from a cultivated tomato plant compared to a control tomato plantcomprising the steps of a) crossing a donor tomato plant with arecipient tomato plant to provide offspring plants, b) quantitativelydetermining the fruit firmness in the fruit of said offspring plants c)establishing a genetic linkage map that links the observed increasedfruit firmness to the presence of at least one DNA marker from saiddonor plant in said offspring plants and d) assigning to a QTL the DNAmarkers on said map that are linked to significantly increased fruitfirmness.

In a specific embodiment, the donor plant has fruit with a significantlyincreased fruit firmness compared to said recipient plant.

The donor plant is preferably S. pennellii and the recipient plant ispreferably S. lycopersicum. In all cases, the recipient plant is thecontrol plant.

The donor plant or the recipient plant could be any one of thefollowing: any plant, line or population within the species Solanumlycopersicum (synonyms are Lycopersicon lycopersicum or Lycopersiconesculentum) or formerly known under the genus name of Lycopersiconincluding but not limited to L. cerasiforrne, L. cheesmanii, L.chilense, L. chmielewskii, L. esculentum (now S. pennellii), L.hirsutum, L. parviborum, L. pennellii, L. peruvianum, L.pimpinellifolium, or S. lycopersicoides. The newly proposed scientificname for L. esculentum is S. pennellii. Similarly, the names of the wildspecies may be altered. L. pennellii has become S. pennellii, L.hirsutum may become S. habrochaites, L. peruvianum may be split into S.‘N peruvianum’ and S. ‘Callejon de Huayles’, S. peruvianum, and S.corneliomuelleri, L. parviflorum may become S. neorickii, L.chmielewskii may become S. chmielewskii, L. chilense may become S.chilense, L. cheesmaniae may become S. cheesmaniae or S. galapagense,and L. pimpinellifolium may become S. pimpinellifolium (Knapp (2005))

In a specific embodiment, the fruit firmness range in offspring plantsis 1.2 to 2.0 times greater than that of fruit produced from a controltomato plant at the harvesting stage.

In a specific embodiment, the fruit firmness range in offspring plantsis 1.2 to 1.5 times greater than that of fruit produced from a controltomato plant at the harvesting stage.

The harvesting stage is preferably the mature green stage.Alternatively, the harvesting stage can be any chosen point in thedevelopment of the tomato fruit, which includes but is not limited tothe immature green stage, rapid expansion stage, mature green stage,breaker stage, red ripe stage or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 daysafter any of these stages, preferably 7 days after the breaker stage.

In a specific embodiment, the at least one DNA marker is found in S.pennellii. Alternatively, the at least one DNA marker could be found inany plant, line or population within the species Solanum lycopersicum(synonyms are Lycopersicon lycopersicum or Lycopersicon esculentum) orformerly known under the genus name of Lycopersicon including but notlimited to L. cerasiforrne, L. cheesmanii, L. chilense, L. chmielewskii,L. esculentum (now S. pennellii), L. hirsutum, L. parviborum, L.pennellii, L. peruvianum, L. pimpinellifolium, or S. lycopersicoides.The newly proposed scientific name for L. esculentum is S. pennellii.Similarly, the names of the wild species may be altered. L. pennelliihas become S. habrochaites, L. hirsutum may become S. pennellii, L.peruvianum may be split into S. ‘N peruvianum’ and S. ‘Callejon deHuayles’, S. peruvianum, and S. corneliomuelleri, L. parviflorum maybecome S. neorickii, L. chmielewskii may become S. chmielewskii, L.chilense may become S. chilense, L. cheesmaniae may become S.cheesmaniae or S. galapagense, and L. pimpinellifolium may become S.pimpinellifolium (Knapp (2005)

There is also provided at least one DNA marker selected from NT3853,NT3907, TG14, HOX7A, CT277, HB2600, TG353, Lm0127, Lm1650, LE5100 andLE5200.

A QTL of the present invention is one or more of a) QTL1 linked to atleast one of the DNA markers NT3853, NT3907 and TG14; or b) QTL2 linkedto at least one of the DNA markers HOX7A and CT277; or c) QTL3 linked toat least one of the DNA markers HB2600, TG353; or d) QTL4 linked to atleast one of the DNA markers Lm0127 and Lm1650; or e) QTL5 linked to atleast one of the DNA markers LE5100 and LE5200.

In a specific embodiment, the QTL is QTL1 linked to at least one of theDNA markers NT3853, NT3907 and TG14; which is present only in the innerpericarp.

In a further aspect there is provided a QTL linked to increased fruitfirmness in fruit provided by a cultivated tomato plant. Preferably theQTL is detected by a method as herein described. Alternatively, the QTLmay be detected by any method known to a person skilled in the art.

Preferably, the at least one QTL of the present invention is present onchromosome 2 of tomato. More preferably, the at least one QTL is presenton chromosome 2 of S. pennellii. More preferably, the at least one QTLis present on the long arm of chromosome 2 of S. pennellii. Mostpreferably, the at least one QTL is present on the long arm ofchromosome 2 of S. pennellii and is any one, two, three, four or all ofQTLs 1 to 5.

In a specific embodiment, the QTL of the present invention is linked toat least one DNA marker selected from the group consisting of NT3853,NT3907, TG14, HOX7A, CT277, HB2600, TG353, Lm0127, Lm1650, LE5100 andLE5200.

The QTL of the present invention is one or more of a) QTL1 linked to atleast one of the DNA markers NT3853, NT3907 and TG14; b) QTL2 linked toat least one of the DNA markers HOX7A and CT277; c) QTL3 linked to atleast one of the DNA markers HB2600, TG353; d) QTL4 linked to at leastone of the DNA markers Lm0127 and Lm1650; e) QTL5 linked to at least oneof the DNA markers LE5100 and LE5200; and combinations thereof.

In a specific embodiment, the QTL is QTL1 linked to at least one of theDNA markers NT3853, NT3907 and TG14; which is present only in the innerpericarp.

There is further provided an isolated DNA sample obtained from a tomatoplant comprising any one, two, three, four or all of QTLs 1 to 5. TheDNA sample may be isolated as described in the examples or by any othermeans familiar to the skilled person.

Production of Tomato Fruit with Increased Firmness by Transgenic Methods

According to another aspect of the presently disclosed subject matter, anucleic acid (in some embodiments a DNA) sequence comprising one or moreof QTLS 1 to 5 or fruit firmness conferring parts thereof, can be usedfor the production of a tomato plant providing fruit with increasedfirmness compared to a control tomato plant. In this aspect, thepresently disclosed subject matter provides for the use of QTLs asdefined herein or increased fruit firmness conferring parts thereof, forproducing a tomato plant providing fruit with increased firmnesscompared to a control tomato plant, which use involves the introductionof a nucleic acid sequence comprising the QTL into a suitable recipientplant. As stated, the nucleic acid sequence can be derived from asuitable donor plant with increased fruit firmness compared to a controltomato plant. A suitable source for the increased fruit firmness locusidentified herein as any of QTLS 1 to 5 is S. pennellii. A number oftomato cultivars that have varying degrees of increased fruit firmnessare commercially available.

The source of the increased fruit firmness loci described herein areintrogression lines IL-2 and IL-3, which were originally generated byDani Zamir and colleagues (Eshed & Zamir, 1994). These lines wereobtained from the Tomato Genetics Resource Center at Davis, Calif.(available at tgrc.ucdavis.edu/) or from Dani Zamir at the HebrewUniversity of Jerusalem, Israel. Once identified in a suitable donorplant, the nucleic acid sequence that comprises a QTL for increasedfruit firmness, or increased fruit firmness—conferring part thereof, canbe transferred to a suitable recipient plant by any method available.For instance, the nucleic acid sequence can be transferred by crossing adonor tomato plant with a recipient plant i.e. by introgression, bytransformation, by protoplast fusion, by a doubled haploid technique, byembryo rescue, or by any other nucleic acid transfer system, followed byselection of offspring plants comprising one or more of the presentlydisclosed QTLs and exhibiting increased fruit firmness. For transgenicmethods of transfer, a nucleic acid sequence comprising a QTL forincreased fruit firmness, or increased fruit firmness—conferring partthereof, can be isolated from the donor plant using methods known in theart, and the thus isolated nucleic acid sequence can be transferred tothe recipient plant by transgenic methods, for instance by means of avector, in a gamete, or in any other suitable transfer element, such asa ballistic particle coated with the nucleic acid sequence.

Plant transformation generally involves the construction of anexpression vector that will function in plant cells. In the presentlydisclosed subject matter, such a vector comprises a nucleic acidsequence that comprises a QTL for increased fruit firmness, or increasedfruit firmness-conferring part thereof, which vector can comprise anincreased fruit firmness conferring gene that is under control of, oroperatively linked to, a regulatory element such as a promoter. Theexpression vector can contain one or more such operably linkedgene/regulatory element combinations, provided that at least one of thegenes contained in the combinations encodes increased fruit firmness.The vector(s) can be in the form of a plasmid, and can be used, alone orin combination with other plasmids, to provide transgenic plants thathave increased fruit firmness using transformation methods known in theart, such as the Agrobacterium transformation system.

The inventors have characterized QTLs 1 to 5 at the molecular level andidentified several putative candidate genes for use in an expressionvector. The list of candidate genes is as follows: QTL1, as describedherein relates to TG451 a MADS-box transcription factor related bysequence to Petunia gene pMADS3 (Tsuchimoto et al. 1993. Plant Cell 5,843-853). QTL2, as described herein relates to the open reading framecorresponding to ethylene responsive transcription factor(SL1.00sc00226_365.1) position 3895565 to 3896029 (−strand). QTL3, asdescribed herein relates to the open reading frame corresponding to apectinesterase/pectinesterase inhibitor (Syngenta GeneChip probe ID,Le0023899). QTL4, as described herein relates to the open reading framecorresponding to dof zinc finger protein 6 (SL1.00sc00226_436.1.1)position 4475868 to 4476845 (+strand). QTL5, as described herein relatesto the open reading frame corresponding to equilibrative nucleosidetransporter family protein (SL1.00sc00226_511.1) position 5133572 to5135660 (−strand). Further details of the ethylene responsivetranscription factor, dof zinc finger 6 and equilibrative nucleosidetransporter protein can be found at solgenomics.net/.

Expression vectors can include at least one marker gene, operably linkedto a regulatory element (such as a promoter) that allows transformedcells containing the marker to be either recovered by negative selection(by inhibiting the growth of cells that do not contain the selectablemarker gene), or by positive selection (by screening for the productencoded by the marker gene). Many commonly used selectable marker genesfor plant transformation are known in the art, and include, for example,genes that code for enzymes that metabolically detoxify a selectivechemical agent that can be an antibiotic or a herbicide, or genes thatencode an altered target which is insensitive to the inhibitor. Severalpositive selection methods are known in the art, such as mannoseselection. Alternatively, marker-less transformation can be used toobtain plants without the aforementioned marker genes, the techniquesfor which are also known in the art.

One method for introducing an expression vector into a plant is based onthe natural transformation system of Agrobacterium (see e.g., Horsch etal., 1985). A. tumefaciens and A. rhizogenes are plant pathogenic soilbacteria that genetically transform plant cells. The Ti and Ri plasmidsof A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant (see e.g., Kado,1991). Methods of introducing expression vectors into plant tissueinclude the direct infection or co-cultivation of plant cells withAgrobacterium tumefaciens (Horsch et al., 1985). Descriptions ofAgrobacterium vectors systems and methods for Agrobacterium-mediatedgene transfer are provided by Gruber & Crosby, 1993, Moloney et al.,1989, and U.S. Pat. No. 5,591,616. General descriptions of plantexpression vectors and reporter genes and transformation protocols anddescriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer can be found in Gruber & Crosby,1993. General methods of culturing plant tissues are provided forexample by Miki et al., 1993 and by Phillips et al., 1988. A referencehandbook for molecular cloning techniques and suitable expressionvectors is Sambrook & Russell, (2001).

Another method for introducing an expression vector into a plant isbased on microprojectile-mediated transformation wherein DNA is carriedon the surface of microprojectiles. The expression vector is introducedinto plant tissues with a ballistic device that accelerates themicroprojectiles to speeds of 300 to 600 m/s which is sufficient topenetrate plant cell walls and membranes (see e.g., Sanford et al.,1987; Klein et al., 1988; Sanford, 1988; Sanford, 1990; Klein et al.,1992; Sanford et al., 1993). Another method for introducing DNA intoplants is via the sonication of target cells (see Zhang et al., 1991).Alternatively, liposome or spheroplast fusion can be used to introduceexpression vectors into plants (see e.g., Deshayes et al., 1985 andChristou et al., 1987). Direct uptake of DNA into protoplasts usingCaCl2 precipitation, polyvinyl alcohol, or poly-L-omithine has also beenreported (see e.g., Hain et al. 1985 and Draper et al., 1982).Electroporation of protoplasts and whole cells and tissues has also beendescribed (D'Halluin et al., 1992 and Laursen et al., 1994).

Other well known techniques such as the use of BACs, wherein parts ofthe tomato genome are introduced into bacterial artificial chromosomes(BACs), ie., vectors used to clone DNA fragments (100- to 300-kb insertsize; average 150 kb) in Escherichia coli cells, based on naturallyoccurring F-factor plasmid found in the bacterium E. coli. (Zhao &Stodoisky, 2004) can be employed for example in combination with the BiBAC system (Hamilton, 1997) to produce transgenic plants. One example ofan over-expression vector is pGWB405 (Nakagawa T, Suzuki T, Murata S atel. Improved Gateway Binary Vectors: High-performance Vectors forCREATION of Fusion Constructs in Transgenic Analysis of Plants.Bioscience biotechnology Biochemistry, 71(8)2095-2010, 2007). Foroverexpression construct production, sequence corresponding to thecomplete open reading frame of the candidate gene involved in modulatingtomato fruit firmness can be cloned in front of the CaMV 35S promoterusing the Gateway clone system which avoids the need for restrictionsites. Such a construct also contains the CaMV terminator at theopposite end. For RNAi construct production, a fragment of the codingsequence unique to the candidate gene involved in modulating tomatofruit firmness can be cloned, for example, in the Gateway system RNAivector pK7GWIWG2(I).

Following transformation of tomato target tissues, expression of theabove described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, using standardregeneration and selection methods.

There is also provided 5 genes the expression levels of which arealtered in recombinants with greater fruit firmness. The 5 genes codefor TG451 (a MADS box transcription factor), ethylene responsivetranscription factor 12, pectin methylesterase, dof zinc finger protein6 and equilibrative nucleoside transporter family protein and were foundto correlate to the positions of QTLs 1 to 5 respectively. mRNAexpression analysis showed that ethylene responsive transcription factor12 was negatively correlated with increased firmness at the breakerstage whereas pectin methylesterase was positively correlated (FIG. 8).Dof zince finger protein mRNA levels were lower in introgression linesIL2-4 and IL2-3 compared with wild type M82 (FIG. 9). In light of thisdata it is predicted that plants, such as tomato plants, which havedownregulated levels of or are absent of ethylene responsivetranscription factor 12 and/or Dof zinc finger protein 6 and plantswhich have upregulated (or overexpressed) levels of pectinmethylesterase would display increased fruit firmness. TG451, a MADS boxtranscription factor, and equilibrative nucleoside transporter familyprotein have also been implicated in improved fruit firmness (seeexample 9).

The present invention therefore provides an isolated nucleotide sequenceselected from the group consisting of: a) a nucleotide sequencecorresponding to the open reading frame or part thereof corresponding tothe following: ethylene responsive transcription factor 12 or pectinmethylesterase or Dof zinc finger protein 6 or TG451 or equilibrativenucleoside transporter family protein); b) a nucleotide sequence that isat least 80% identical to the nucleotide sequence of a); c) a nucleotidesequence comprising at least 21 consecutive nucleotides of thenucleotide sequence of a); d) a nucleotide sequence that hybridisesunder stringent conditions to the complement of any of nucleotidesequences a) to c); and e) a nucleotide sequence that is the complementto the nucleotide sequences of any one of a) to d). In one embodiment,the nucleotide sequence of step b) is at least 90% identical to thenucleotide sequence of a).

In one embodiment, the isolated nucleotide sequence of the invention isTG451, a MADS box transcription factor. In another embodiment, theisolated nucleotide sequence of the invention is ethylene responsivetranscription factor 12. In another embodiment, the isolated nucleotidesequence of the invention is pectin methylesterase. In anotherembodiment, the isolated nucleotide sequence of the invention is Dofzinc finger protein 6. In another embodiment, the isolated nucleotidesequence of the invention is equilibrative nucleoside transporter familyprotein.

There is also provided a vector comprising the isolated nucleotidesequence of the invention. In one embodiment, the isolated nucleotidesequence is in the sense orientation. In another embodiment, theisolated nucleotide sequence is in the antisense orientation.

There is also provided a host cell which expresses a vector of theinvention.

There is also provided a transgenic plant or part thereof comprising ahost cell of the invention. In one embodiment, the transgenic plant orpart thereof is a monocot. In another embodiment, the plant or partthereof is a dicot, for example a tomato.

There is also provided a method for producing a transgenic plantcomprising regenerating a plant from a host cell according to theinvention.

There is also provided a cultivated plant or part thereof produced by amethod according to the invention.

There is also provided a method of manipulating the texture of fruit ofa transgenic plant, for example a tomato plant comprising transformingsaid plant with a vector of the invention. In one embodiment, the speedof fruit ripening is increased when compared with fruit from anuntransformed plant. In another embodiment, the speed of fruit ripeningis decreased when compared with fruit from an untransformed plant. Inone embodiment, the speed of tomato ripening is measured at breakerstage.

There is also provided a method of manipulating tomato fruit pigmentcontent in fruit of a transgenic plant, for example a tomato plantcomprising transforming said plant with a vector of the invention. Inone embodiment, the fruit pigment content is increased compared withfruit from an untransformed plant. In one embodiment, the fruit pigmentcontent is decreased compared with fruit from an untransformed plant. Inone embodiment, the fruit pigment content is measured at breaker stage.

There is also provided a plant, for example a tomato plant or partthereof obtained by a method of the invention.

There is also provided a method of detecting genetic markers indicativeof tomato texture of a plant of the Solanaceae family, comprisingisolating DNA from said plant and from one or both parents of saidplant; screening for genetic markers in a region of said DNA at or nearsequence corresponding to an isolated sequence of the invention; anddetermining co-inheritance of said markers from one or both parents tosaid individual.

There is also provided a genetic marker detectable by a method ofdetecting genetic markers of the invention.

There is also provided use of a genetic marker of the invention for theproduction of a cultivated tomato plant capable of bearing tomato fruit.

There is also provided a cultivated tomato plant or part thereofproduced by a method of the invention.

There is also provided use of a cultivated tomato plant or part thereofaccording to the invention in the fresh cut market or for foodprocessing.

There is also provided use of an isolated nucleotide sequence of theinvention in the manipulation of speed of ripening or of pigment contentof fruit of a plant, preferably a tomato plant, wherein saidmanipulation is effected by genetic modification of said plant.

There is also provided use of a method according to the invention,wherein said genetic modification is introduced by a method selectedfrom the list consisting of transposon insertion mutagenesis, T-DNAinsertion mutagenesis, TILLING, site-directed mutagenesis, directedevolution, and homologous recombination. In one embodiment, geneticmodification is introduced by TILLING.

Production of Tomato Plants with Increased Fruit Firmness by NonTransgenic Methods

In some embodiments for producing a tomato plant with increased fruitfirmness, protoplast fusion can be used for the transfer of nucleicacids from a donor plant to a recipient plant. Protoplast fusion is aninduced or spontaneous union, such as a somatic hybridization, betweentwo or more protoplasts (the cell walls of which are removed byenzymatic treatment) to produce a single bi- or multi-nucleate cell. Thefused cell, which can even be obtained with plant species that cannot beinterbred in nature, is tissue cultured into a hybrid plant exhibitingthe desirable combination of traits. More specifically, a firstprotoplast can be obtained from a tomato plant or other plant line thatexhibits increased fruit firmness. A second protoplast can be obtainedfrom a second tomato plant or other plant variety, preferably a tomatoplant line that comprises commercially valuable characteristics. Theprotoplasts are then fused using traditional protoplast fusionprocedures, which are known in the art.

Alternatively, embryo rescue can be employed in the transfer of anucleic acid comprising one or more QTLs as described herein from adonor tomato plant to a recipient tomato plant. Embryo rescue can beused as a procedure to isolate embryos from crosses wherein plants failto produce viable seed. In this process, the fertilized ovary orimmature seed of a plant is tissue cultured to create new plants(Pierik, 1999). The presently disclosed subject matter also relates tomethods for producing tomato plant with increased fruit firmnesscomprising performing a method for detecting the presence of a QTLlinked to increased fruit firmness in a donor tomato plant as describedherein, and transferring a nucleic acid sequence comprising at least oneQTL thus detected, or an increased fruit firmness-conferring partthereof, from the donor plant to a recipient tomato plant. The transferof the nucleic acid sequence can be performed by any of the methodspreviously described herein.

An exemplary embodiment of such a method comprises the transfer byintrogression of the nucleic acid sequence from a donor tomato plantinto a recipient tomato plant by crossing the plants. This transfer canthus suitably be accomplished by using traditional breeding techniques.QTLs are introgressed in some embodiments into commercial tomatovarieties using marker-assisted selection (MAS) or marker-assistedbreeding (MAB). MAS and MAB involves the use of one or more of themolecular markers for the identification and selection of thoseoffspring plants that contain one or more of the genes that encode forthe desired trait. In the context of the presently disclosed subjectmatter, such identification and selection is based on selection of QTLsof the presently disclosed subject matter or markers associatedtherewith. MAS can also be used to develop near-isogenic lines (NIL)harboring the QTL of interest, allowing a more detailed study of eachQTL effect and is also an effective method for development of backcrossinbred line (BIL) populations (see e.g., Nesbitt & Tanksley, 2001; vanBerloo et al., 2001). Tomato plants developed according to theseembodiments can advantageously derive a majority of their traits fromthe recipient plant, and derive increased fruit firmness from the donorplant. As discussed herein, traditional breeding techniques can be usedto introgress a nucleic acid sequence encoding for increased fruitfirmness into a recipient tomato plant. In some embodiments, a donortomato plant that exhibits increased fruit firmness and comprising anucleic acid sequence encoding for increased fruit firmness is crossedwith a recipient tomato plant that in some embodiments exhibitscommercially desirable characteristics.

The resulting plant population (representing the F1 hybrids) is thenself-pollinated and set seeds (F2 seeds). The F2 plants grown from theF2 seeds are then screened for increased fruit firmness by methods knownto the skilled person.

Tomato plant lines with increased fruit firmness can be developed usingthe techniques of recurrent selection and backcrossing, selfing, and/ordihaploids, or any other technique used to make parental lines. In amethod of recurrent selection and backcrossing, increased fruit firmnesscan be introgressed into a target recipient plant (the recurrent parent)by crossing the recurrent parent with a first donor plant, which differsfrom the recurrent parent and is referred to herein as the“non-recurrent parent”. The recurrent parent is a plant that does nothave increased fruit firmness but does possess commercially desirablecharacteristics.

In some embodiments, the non-recurrent parent exhibits increased fruitfirmness and comprises a nucleic acid sequence that encodes forincreased fruit firmness. The non-recurrent parent can be any plantvariety or inbred line that is cross-fertile with the recurrent parent.

The progeny resulting from a cross between the recurrent parent andnon-recurrent parent are backcrossed to the recurrent parent. Theresulting plant population is then screened for increased fruitfirmness. Marker-assisted selection (MAS) can be performed using one ormore of the herein described molecular markers, or by usinghybridization probes, or polynucleotides to identify those progeny thatcomprise a nucleic acid sequence encoding for increased fruit firmness.Also, MAS can be used to confirm the results obtained from thequantitative fruit firmness measurements.

Following screening, the F1 hybrid plants that exhibit an increasedfruit firmness phenotype or, in some embodiments, genotype and thuscomprise the requisite nucleic acid sequence encoding for increasedfruit firmness, are then selected and backcrossed to the recurrentparent for a number of generations in order to allow for the tomatoplant to become increasingly inbred. This process can be performed fortwo, three, four, five, six, seven, eight, or more generations. Inprinciple, the progeny resulting from the process of crossing therecurrent parent with the increased fruit firmness non-recurrent parentare heterozygous for one or more genes that encode for increased fruitfirmness.

In general, a method of introducing a desired trait into a hybrid tomatovariety may comprise:

(a) crossing an inbred tomato parent with another tomato plant thatcomprises one or more desired traits, to produce F1 progeny plants,wherein the desired trait is increased fruit firmness;

(b) selecting the F1 progeny plants that have the desired trait toproduce selected F1 progeny plants, in some embodiments using molecularmarkers as described herein;

(c) backcrossing the selected progeny plants with the inbred tomatoparent plant to produce backcross progeny plants;

(d) selecting for backcross progeny plants that have the desired traitand morphological and physiological characteristics of the inbred tomatoparent plant, wherein the selection comprises the isolation of genomicDNA and testing the DNA for the presence of at least one molecularmarker for QTL1, QTL2, QTL3, QTL4 and/or QTL5, in some embodiments asdescribed herein;(e) repeating steps (c) and (d) two or more times in succession toproduce selected third or higher backcross progeny plants;(f) optionally selfing selected backcross progeny in order to identifyhomozygous plants; and(g) crossing at least one of the backcross progeny or selfed plants withanother inbred tomato parent plant to generate a hybrid tomato varietywith the desired trait and all of the morphological and physiologicalcharacteristics of hybrid tomato variety when grown in the sameenvironmental conditions.

As indicated, the last backcross generation can be selfed in order toprovide for homozygous pure breeding (inbred) progeny having increasedfruit firmness. Thus, the result of recurrent selection, backcrossing,and selfing is the production of lines that are genetically homogenousfor the genes linked to increased fruit firmness, and in someembodiments as well as for other genes linked to traits of commercialinterest.

Accordingly, there is provided a method of producing a tomato plantwhich provides fruit with increased fruit firmness as herein described.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described comprising the steps of performing a method fordetecting a QTL linked to increased fruit firmness as herein described,and transferring a nucleic acid comprising at least one QTL thusdetected, from a donor tomato plant to a recipient tomato plant, whereinsaid increased fruit firmness is measured in fruit from an offspringcultivated tomato plant compared to fruit from a control tomato plant.

In a specific embodiment there is provided a method of producing acultivated tomato plant which provides fruit with increased fruitfirmness as herein described wherein said transfer of nucleic acid isperformed by transformation, by protoplast fusion, by a doubled haploidtechnique or by embryo rescue.

In a specific embodiment there is provided a method of producing acultivated tomato plant which provides fruit with increased fruitfirmness as herein described, wherein the fruit firmness range in theoffspring tomato plant is 1.2 to 2.0 times greater than fruit of acontrol tomato plant at the harvesting stage. Alternatively, the fruitfirmness range in the offspring tomato plant is 1.2 to 1.5 times greaterthan fruit from a control tomato plant at the harvesting stage.Preferably the harvesting stage is mature green stage. Alternatively,the harvesting stage can be the immature green stage, rapid expansionstage, breaker stage or red ripe stage or 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 days after any of these stages.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described, wherein the fruit firmness range remains up untilbreaker plus 7 days.

In a further aspect there is provided a method of producing a tomatoplant which provides fruit with increased fruit firmness as hereindescribed, wherein the donor plant is S. pennellii and the recipientplant is S. lycopersicum. Alternatively, the donor plant or therecipient plant could be any one of the following: any plant, line orpopulation within the species Solanum lycopersicum (synonyms areLycopersicon lycopersicum or Lycopersicon esculentum) or formerly knownunder the genus name of Lycopersicon including but not limited to L.cerasiforrne, L. cheesmanii, L. chilense, L. chmielewskii, L. esculentum(now S. pennellii), L. hirsutum, L. parviborum, L. pennellii, L.peruvianum, L. pimpinellifolium, or S. lycopersicoides. The newlyproposed scientific name for L. esculentum is S. pennellii. Similarly,the names of the wild species may be altered. L. pennellii has become S.pennellii, L. hirsutum may become S. habrochaites, L. peruvianum may besplit into S. ‘N peruvianum’ and S. ‘Callejon de Huayles’, S.peruvianum, and S. corneliomuelleri, L. parviflorum may become S.neorickii, L. chmielewskii may become S. chmielewskii, L. chilense maybecome S. chilense, L. cheesmaniae may become S. cheesmaniae or S.galapagense, and L. pimpinellifolium may become S. pimpinellifolium(Knapp (2005).

There is also provided a method of producing a tomato plant whichprovides fruit with increased fruit firmness as herein described,wherein said QTL is one or more of a) QTL1 linked to at least one of theDNA markers NT3853, NT3907 and TG14; or b) QTL2 linked to at least oneof the DNA markers HOX7A and CT277; or c) QTL3 linked to at least one ofthe DNA markers HB2600, TG353; d) QTL4 linked to at least one of the DNAmarkers Lm0127 and Lm1650; e) QTL5 linked to at least one of the DNAmarkers LE5100 and LE5200.

There is also provided a method of producing a tomato plant whichprovides fruit with increased fruit firmness as herein described,wherein said QTL is QTL1 linked to at least one of the DNA markersNT3853, NT3907 and TG14; which is present only in the inner pericarp.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described, wherein said QTLs are QTL1 linked to at least one ofthe DNA markers NT3853, NT3907 and TG14; and QTL2 linked to at least oneof the DNA markers HOX7A and CT277.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described, wherein said QTLs are QTL1 linked to at least one ofthe DNA markers NT3853, NT3907 and TG14; and QTL3 linked to at least oneof the DNA markers HB2600, TG353.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described, wherein said QTLs are QTL1 linked to at least one ofthe DNA markers NT3853, NT3907 and TG14; and QTL4 linked to at least oneof Lm0127 and Lm1650.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described, wherein said QTLs are QTL1 linked to at least one ofthe DNA markers NT3853, NT3907 and TG14; and QTL5 linked to at least oneof LE5100 and LE5200.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described, wherein said QTLs are QTL2 linked to at least one ofthe DNA markers HOX7A and CT277; and QTL3 linked to at least one of theDNA markers HB2600, TG353.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described, wherein said QTLs are QTL2 linked to at least one ofthe DNA markers HOX7A and CT277; and QTL4 linked to at least one ofLm0127 and Lm1650.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described, wherein said QTLs are QTL2 linked to at least one ofthe DNA markers HOX7A and CT277; and QTL5 linked to at least one ofLE5100 and LE5200.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described, wherein said QTLs are QTL3 linked to at least one ofthe DNA markers HB2600 and TG353; and QTL4 linked to at least one ofLm0127 and Lm1650.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described, wherein said QTLs are QTL3 linked to at least one ofthe DNA markers HB2600 and TG353; and QTL5 linked to at least one ofLE5100 and LE5200.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described, wherein said QTLs are QTL4 linked to at least one ofLm0127 and Lm1650; and QTL5 linked to at least one of LE5100 and LE5200.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described, wherein said QTLs are QTL1 linked to at least one ofthe DNA markers NT3853, NT3907 and TG14; and QTL2 linked to at least oneof the DNA markers HOX7A and CT277; and QTL3 linked to at least one ofthe DNA markers HB2600, TG353; and QTL4 linked to at least one of Lm0127and Lm1650; and QTL5 linked to at least one of LE5100 and LE5200.

In a specific embodiment there is provided a method of producing atomato plant which provides fruit with increased fruit firmness asherein described, wherein said QTL is QTL1 linked to at least one of theDNA markers NT3853, NT3907 and TG14; which is present only in the innerpericarp.

In a further aspect there is provided a tomato plant, or part thereof,obtainable by a method as herein described.

In a further aspect there is provided a cultivated tomato plantcomprising a QTL responsible for increased fruit firmness as hereindescribed.

In a further aspect there is provided a hybrid tomato plant, or partthereof, obtainable by crossing a tomato plant as herein described witha tomato plant that exhibits commercially desirable characteristics.

In a further aspect there is provided a tomato seed produced by growingthe tomato plant as herein described.

In a further aspect there is provided a tomato seed produced by crossingthe cultivated tomato plant as herein described with a plant havingdesirable phenotypic traits to obtain a plant that has significantlyincreased fruit firmness compared to a control plant.

In a further aspect there is provided use of a QTL as herein describedfor the production of tomato plants having increased fruit firmnesscompared to control plants.

In a further aspect there is provided use of a tomato plant havingincreased fruit firmness as herein described for expanding theharvesting slot of tomato fruit. The harvesting slot can be expanded byany one of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

In a further aspect there is provided use of a tomato plant havingincreased fruit firmness as herein described in the fresh cut market orfor food processing.

In a further aspect there is provided processed food made from a tomatoplant comprising the at least one QTL as herein described.

Embodiments of the Invention

Embodiment 1: a tomato fruit with significantly increased fruit firmnessat the harvesting stage linked to at least one genetic element in thecultivated tomato plant producing said tomato fruit, wherein saidfirmness is from between 1.2 to 2.0 times greater than that of fruitfrom a control tomato plant which does not have the said at least onegenetic element.

Embodiment 2: a tomato fruit according to embodiment 1 wherein theharvesting stage is the mature green stage

Embodiment 3: a tomato fruit according to embodiment 1 or 2 wherein thefruit firmness is from between 1.2 to 1.5 times greater than thatproduced from a control tomato plant which does not have the said atleast one genetic element.

Embodiment 4: a tomato fruit according to any one of embodiments 1 to 3,wherein said firmness range is measured at breaker plus 7 days.

Embodiment 5: a tomato fruit according to any preceding embodimentwherein the at least one genetic element is located on the long arm ofchromosome 2.

Embodiment 6: a cultivated tomato plant which produces tomato fruitaccording to any of embodiments 1 to 5 wherein said plant can becharacterised by a) the at least one genetic element is linked to atleast one DNA marker selected from the group consisting of NT3853,NT3907, TG14, HOX7A, CT277, HB2600, TG353, Lm0127, Lm1650, LE5100 andLE5200 and/or b) the at least one genetic element is complementary tothe corresponding genetic element in Solanum pennellii lines IL2-3 andIL2-4 deposited under accession numbers LA4038 and LA4039, respectively,wherein the said at least one genetic element in LA4038 and LA4039 islinked to at least one DNA marker selected from the group consisting ofNT3853, NT3907, TG14, HOX7A, CT277, HB2600, TG353, Lm0127, Lm1650,LE5100 and LE5200.

Embodiment 7: a cultivated tomato plant according to embodiment 6wherein the at least one genetic element is one or more QTL selectedfrom a) QTL1 linked to at least one of the DNA markers NT3853, NT3907and TG14; or b) QTL2 linked to at least one of the DNA markers HOX7A andCT277; or c) QTL3 linked to at least one of the DNA markers HB2600,TG353; or d) QTL4 is linked to at least one of the DNA markers Lm0127and Lm1650; or e) QTL5 is linked to at least one of the DNA markersLE5100 and LE5200.

Embodiment 8: a cultivated tomato plant according to embodiment 7wherein the QTL is QTL1 associated with at least one of the DNA markersNT3853, NT3907 and TG14; which is present only in the inner pericarp.

Embodiment 9: a cultivated tomato plant according to any one ofembodiments 6 to 8 wherein said plant is an inbred, a dihaploid or ahybrid.

Embodiment 10: a cultivated tomato plant according to embodiment 9wherein said plant is male sterile.

Embodiment 11: a tomato seed which produces a cultivated tomato plantaccording to any one of embodiments 6 to 10

Embodiment 12: Plant part of a cultivated tomato plant according toembodiment 11.

Embodiment 13: Plant material obtainable from a plant part of acultivated tomato plant according to embodiment 12.

Embodiment 14: a method for detecting a QTL linked to significantlyincreased fruit firmness in fruit from a cultivated tomato plantcompared to a control tomato plant comprising the steps of a) crossing adonor tomato plant with a recipient tomato plant to provide offspringcultivated tomato plants, b) quantitatively determining the firmness inthe fruit of said offspring plants c) establishing a genetic linkage mapthat links the observed increased fruit firmness to the presence of atleast one DNA marker from said donor plant in said offspring plants andd) assigning to a QTL the DNA markers on said map that are linked tosignificantly increased fruit firmness.

Embodiment 15: The method according to embodiment 14 wherein said donorplant has a significantly increased fruit firmness compared to saidrecipient plant.

Embodiment 16: The method of embodiment 14 or 15 wherein the donor plantis Solanum pennellii and the recipient plant is Solanum lycopersicum.

Embodiment 17: The method of any one of embodiments 14 to 16 wherein thefruit firmness range in offspring plants is 1.2 to 2.0 times greaterthan that of fruit produced from a control tomato plant at theharvesting stage.

Embodiment 18: The method of embodiment 17 wherein the fruit firmnessrange in offspring plants is 1.2 to 1.5 times greater than that of fruitproduced from a control tomato plant at the harvesting stage.

Embodiment 19: The method of any one of embodiments 17 to 18 wherein theharvesting stage is the mature green stage.

Embodiment 20: The method of any one of embodiments 14 to 19 wherein theat least one DNA marker is found in Solanum pennellii.

Embodiment 21: The method of any one of embodiments 14 to 20 wherein theat least one DNA marker is selected from NT3853, NT3907, TG14, HOX7A,CT277, HB2600, TG353, Lm0127, Lm1650, LE5100 and LE5200.

Embodiment 22: The method of any one of embodiments 14 to 21 whereinsaid QTL is one or more of a) QTL1 linked to at least one of the DNAmarkers NT3853, NT3907 and TG14; or b) QTL2 linked to at least one ofthe DNA markers HOX7A and CT277; or c) QTL3 linked to at least one ofthe DNA markers HB2600, TG353; or d) QTL4 linked to at least one of theDNA markers Lm0127 and Lm1650; or e) QTL5 linked to at least one of theDNA markers LE5100 and LE5200.

Embodiment 23: The method of embodiment 22 wherein said QTL is QTL1associated with at least one of the DNA markers NT3853, NT3907 and TG14;which is present only in the inner pericarp.

Embodiment 24: a QTL responsible for increased fruit firmness in fruitprovided by a cultivated tomato plant detected by a method according toany one of embodiments 14 to 23.

Embodiment 25: a QTL according to embodiment 24 located on the long armof chromosome 2.

Embodiment 26: a QTL according to any one of embodiments 24 to 25associated with at least one DNA marker selected from the groupconsisting of NT3853, NT3907, TG14, HOX7A, CT277, HB2600, TG353, Lm0127,Lm1650, LE5100 and LE5200.

Embodiment 27: a QTL according to any one of embodiments 24 to 26wherein said QTL is one or more of a) QTL1 linked to at least one of theDNA markers NT3853, NT3907 and TG14; b) QTL2 linked to at least one ofthe DNA markers HOX7A and CT277; c) QTL3 linked to at least one of theDNA markers HB2600, TG353; d) QTL4 linked to at least one of the DNAmarkers Lm0127 and Lm1650; e) QTL5 linked to at least one of the DNAmarkers LE5100 and LE5200.

Embodiment 28: a QTL according to embodiment 27 wherein said QTL is QTL1linked to at least one of the DNA markers NT3853, NT3907 and TG14; whichis present only in the inner pericarp.

Embodiment 29: An isolated DNA sample obtained from a tomato plantcomprising a QTL according to any one of embodiments 24 to 28.

Embodiment 30: a method of producing a cultivated tomato plant whichprovides fruit with significantly increased fruit firmness according toany one of embodiments 1 to 5

Embodiment 31: a method of producing a cultivated tomato plant whichprovides fruit with increased fruit firmness according to embodiment 30comprising the steps of performing a method for detecting a QTLassociated with significantly increased fruit firmness according to anyone of embodiments 14 to 23, and transferring a nucleic acid comprisingat least one QTL thus detected, from a donor tomato plant to a recipienttomato plant, wherein said increased fruit firmness is measured in fruitfrom an offspring cultivated tomato plant compared to fruit from acontrol tomato plant.

Embodiment 32: a method of producing a cultivated tomato plant whichprovides fruit with increased fruit firmness according to any one ofembodiments 30 to 31 wherein said transfer of nucleic acid is performedby transformation, by protoplast fusion, by a doubled haploid techniqueor by embryo rescue.

Embodiment 33: a method of producing a cultivated tomato plant whichprovides fruit with increased fruit firmness according to any one ofembodiments 30 to 32, wherein the fruit firmness range in the donortomato plant is 1.2 to 2.0 times greater than fruit of a control tomatoplant at the mature green stage.

Embodiment 34: a method of producing a cultivated tomato plant whichprovides fruit with increased fruit firmness according to any one ofembodiments 30 to 33, wherein the fruit firmness range is measured atbreaker plus 7 days.

Embodiment 35: a method of producing a cultivated tomato plant whichprovides fruit with increased fruit firmness according to any one ofembodiments 31 to 34, wherein the donor plant is Solanum pennellii andthe recipient plant is Solanum lycopersicum.

Embodiment 36: a method of producing a cultivated tomato plant whichprovides fruit with increased fruit firmness according to any one ofembodiments 31 to 35 wherein said QTL is one or more of a) QTL1 linkedto at least one of the DNA markers NT3853, NT3907 and TG14; or b) QTL2linked to at least one of the DNA markers HOX7A and CT277; or c) QTL3linked to at least one of the DNA markers HB2600, TG353; or d) QTL4linked to at least one of the DNA markers Lm0127 and Lm1650; or e) QTL5linked to at least one of the DNA markers LE5100 and LE5200.

Embodiment 37: a method of producing a cultivated tomato plant whichprovides fruit with increased fruit firmness according to embodiment 36wherein said QTL is QTL1 linked to at least one of the DNA markersNT3853, NT3907 and TG14; which is present only in the inner pericarp.

Embodiment 38: a cultivated tomato plant, or part thereof, obtainable bya method according to any one of embodiments 30 to 37.

Embodiment 39: a cultivated tomato plant comprising a QTL responsiblefor increased fruit firmness according to any one of embodiments 24 to28.

Embodiment 40: a hybrid tomato plant, or part thereof, obtainable bycrossing a cultivated tomato plant according to embodiments 6 to 10 and38 to 39 with a tomato plant that exhibits commercially desirablecharacteristics.

Embodiment 41: Tomato seed produced by growing the tomato plant of anyone of embodiments 6 to 10 and 38 to 40.

Embodiment 42: Tomato seed produced by crossing the cultivated tomatoplant of any one of embodiments 6 to 10 and 38 to 40 with a plant havingdesirable phenotypic traits to obtain a plant that has significantlyincreased fruit firmness compared to a control plant.

Embodiment 43: Use of a QTL according to any one of embodiments 24 to 28for the production of tomato plants having significantly increased fruitfirmness compared to control tomato plants.

Embodiment 44: Use of a tomato plant according to any one of embodiments6 to 10 and 38 to 40 for expanding the harvesting slot of tomato fruit.

Embodiment 45: Use of a tomato plant according to any one of embodiments6 to 10 and 38 to 40 in the fresh cut market or for food processing.

Embodiment 46: Use of a tomato fruit according to any one of embodiments1 to 5 in the fresh cut market or for food processing

Embodiment 47: Processed food made from a tomato plant comprising the atleast one genetic element according to embodiments 6 to 10.

Deposited Lines

The following seed samples were deposited with NCIMB, Ferguson Building,Craibstone Estate, Bucksbum, Aberdeen AB21 9YA, Scotland, UK, on Oct.22, 2009 under the provisions of the Budapest Treaty in the name ofSyngenta Participations AG:

Solanum lycopersicum CV M82, deposit number NCIMB 41661

Solanum lycopersicum QTL NIL 301, deposit number NCIMB 41662

Line details: F2 recombinants of line IL2-3 and F3 of IL2-4 were testedfor increased fruit firmness. Extra firm lines were identified includingQTL recombinant line 301. F3 seed this heterozygous recombinant 301 weresown and homozygous individuals identified using markers. The F4 seedwere collected and five lines re-grown and leaf DNA checked withmarkers. The F5 seed were then collected and deposited as QTL-NIL seedfrom line 301. Seed from CV M82 as the cultivated parent have also beendeposited.

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EXAMPLES Example 1 Fruit Firmness Measurements of Introgression Lines

Seventy six S. pennellii ILs were grown in summer 2006 under standardgreenhouse conditions (as described in the “Definitions” section). Tomeasure fruit firmness, a transverse section was cut from the centre ofthe fruit using a double blade mounted on a customized cutting tool togive a section of exactly 6 mm. This section was placed on a flat plate.Using a Lloyd LRX machine equipped with a 10 N load cell, the forcerequired to penetrate the outer and inner pericarp tissue by a 6 mmprobe travelling at 10 mm/minute (maximum load) was then measured. Thesefruit firmness measurements on ripening fruit demonstrated that IL2-3and IL2-4 lines had significantly increased fruit firmness duringripening in comparison with the M82 control. The most pronounced effectswere in IL2-3 and fruit firmness data from the outer and inner pericarpof this line is shown in Tables 1 and 2 below.

In each table, firmness is represented by measures of maximum load (N).The values are predicted means for lines in 2 years (2007 and 2008) asgenerated using the Residual Maximum Likelihood (REML) statistic andtakes into account variation resulting from fruit weight, truss number,date of harvest and position in glasshouse.

TABLE 1 Outer Pericarp Line Year 1 (2007) Year 2 (2008 ) M82 cultivated0.56 ± 0.05 0.38 ± 0.04 parent n = 423 n = 122 IL2-3 S. pennellii 1.09 ±0.07 1.35 ± 0.08 introgression line n = 53 n = 26 301 (IL2-3 2.61 ± 0.141.31 ± 0.13 recombinant*) n = 11 n = 10 142 (IL2-3 0.37 ± 0.14 0.38 ±0.13 recombinant*) n = 10 n = 8

TABLE 2 Inner Pericarp Line Year 1 (2007) Year 2 (2008 ) M82 cultivated0.42 ± 0.04 0.39 ± 0.05 parent n = 423 n = 122 IL2-3 S. pennellii 0.66 ±0.07 2.23 ± 0.09 introgression line n = 53 n = 26 301 (IL2-3 1.38 ± 0.131.47 ± 0.14 recombinant*) n = 11 n = 10 142 (IL2-3 0.31 ± 0.14 0.34 ±0.15 recombinant*) n = 10 n = 8 *These lines were used with many othersto generate the QTL maps. They are F2 lines and hence still heterozygousat some loci.

Example 2 Development of Markers to Delineate Recombination Events inthe IL2-3 and IL2-4 Introgression Lines and Confirmation of GenotypesUsed in Experiments

Based on the tomato genetic map (Tanksley et al, 1992;www.sgn.cornell.edu), markers were selected (FIG. 1) to distinguish anddelimit the 2-3 and 2-4 introgressions on chromosome 2. Singlenucleotide polymorphisms (SNPs) were identified between S. lycopersicumand S. pennellii. These were used to verify the genotype of all 7200lines used in the experiments. Taqman-based markers for CT255, TG353,TG451 and TG583 (FIG. 1) were then generated to allow high throughputscreening for informative recombinants. FIG. 1 is a cartoon showingmarkers delineating (A) IL2-3 (B) IL2-4 and (C) QTL-NIL 301.

Example 3 Establishment of Whether the Increased Fruit Firmness Trait isDominant or Recessive and Collection of Sufficient IL2-3×M82F2 andIL2-4×M82F2 Seed to be Grown for Screening for Recombination Events

It has been shown that the S. pennellii fruit firmness alleles aredominant over the M82 allele because fruit from the F1 line for thebackcross between IL2-3 and M82 the fruit were firmer than the M82parent. Seeds from crosses between IL2-3×M82 and IL2-4×M82 were obtainedfrom Dani Zamir (The Hebrew University of Jerusalem). In most cases F1seed was grown, but in some cases this was not available and F2 seed wasobtained by selfing and selection of heterozygous F2 individuals. Seedfrom these individuals was collected to screen for recombinants.

The ripening fruit of these F1 and F2 heterozygous lines were thentested for mechanical properties after growth under standard conditions.Fruits were collected at 7 days after the first sign of red colour (daypost breaker). A transverse section was cut from the centre of the fruitusing a double blade mounted on a customized cutting tool to give asection of exactly 6 mm. This section was placed on a flat plate. Usinga Lloyd LRX machine equipped with a 10 N load cell, the force requiredto penetrate the outer and inner pericarp tissue by a 6 mm probetravelling at 10 mm/minute (maximum load) was then measured. Thismeasurement was repeated 3 times for each pericarp layer on each fruit.Maximum load is the maximum resistance achieved whilst the probe ispassing through the tissue. At least 5 fruit were taken from each lineat each stage of development unless otherwise stated. The fruit of theIL2-3 parental line and those of the IL2-3 F1 lines retained significantfirmness during the later stages of ripening in comparison with the M82controls. For example, in comparison with M82, in IL2-3 F1 (FIG. 2,panels A+B), at B+5 and B+7 (the stage at which the fruit would beconsumed) the outer pericarp was significantly (P<0.01) firmer at theequivalent stage of ripening. For the IL2-4 lines differences in fruitfirmness were also apparent in comparison to M82 but low numbers offruit due to blossom end rot in this line impeded proper statisticalanalysis. An additional IL for Chromosome 2 (IL2-3-1) was obtained fromDani Zamir. This covered just the upper end of the 2-3 introgression(corresponding to flanking markers CT176 and TG554), above the overlapbetween IL2-3 and 2-4. Fruits from this line had similar fruit firmnesscharacteristics (i.e. maximum load) or were softer than M82 at all thestages of ripening (data not shown).

Fruits from all lines ripened to a fully red colour at B+7 (ie all thesurface of the pericarp was red and this was also the case for internalpericarp tissues).

Example 4 Screening Seedlings for Recombination Events, SelectingRecombinants and Fruit Firmness Testing 10 Individual Fruit/Lines

1200 IL2-3×M82F2 and IL2-4×M82F3 seedlings were grown under standardgrowth conditions and DNA was extracted using standard methods from thefirst true leaf. Recombination events were screened for using bothintrogressions because of their large overlap in this region likely toharbour the QTL. Taqman-based markers were used to identify recombinantindividuals. From the first 1200 lines, 89 recombinants were identified.One of these plants failed to produce a meristem and died. The remainingplants were rescreened through sequencing the SNPs and the genotypes of78 individuals were verified. Ten lines scored as heterozygous by Taqmancould not be confirmed by the SNP assay.

Example 5 Genotyping Recombinant Lines

The 78 recombinant individuals were then genotyped with a further 38markers on the Syngenta SSR platform. Markers were ordered using JoinMap and then displayed in Microsoft Excel with genotype data for eachline. Lines shown were selected to show a variety of the recombinantsobtained (FIG. 3). These markers spanned the IL2-3 and 2-4introgressions. These markers have been set more precisely within thecontext of the tomato genetic map by genotyping the recombinants withseveral additional SNP markers derived from RFLP resources (see QTL mapin FIG. 7, panels A+B). The SSR platform reveals a random distributionof recombination events. Therefore with 78 recombinants across a 50 cMinterval, substantial resolution of the fruit firmness QTL has beenachieved.

Example 6 Establishment of a Mapping Interval of Less than 5Centimorgans for Fruit Firmness QTL

Fruit firmness measurements on 10 individual fruits at breaker+7 daysfrom each of the 78 recombinant lines were made as described above. Aregion of less than 5 centimorgans (<5 cM) on which to focus positionalcloning experiments was then delineated (FIG. 4, panels A+B). Markersflanking this region were developed from the tomato genetic map, genomesequence data and from the Syngenta ultra-high density map as necessary.These were then used to screen a further 6000 F2 seedlings forinformative recombinants in the mapping interval (see example 8).

QTL1 is only present at breaker+7 days in the inner pericarp and not theouter pericarp (FIG. 4). QTL analysis was undertaken using several wellestablished statistical packages including MapQTL and QTL Cartographerwith essentially identical results: (van Ooijen J W, Maliepaard C (1996)MapQTL® version 4.0: Software for the calculation of QTL positions ongenetic maps. CPRO-DLO, Wageningen; van Oijen J W, Voorips RE (2001)JoinMap® 3.0: Software for the calculation of genetic linkage maps.Plant Research International, Wageningen, Netherlands; Wang S., C. J.Basten, and Z.-B. Zeng (2007). Windows QTL Cartographer 2.5 Departmentof Statistics, North Carolina State University, Raleigh, N.C. (availableat statgen.ncsu.edu/qtlcart/WQTLCart.htm).

In certain sub-lines harbouring the major fruit firmness QTLs 1 to 5substantial additive effects on fruit firmness were observed above thoseapparent in the IL parents (FIG. 5). These differences in fruit firmnessare large and highly significant (P<0.001).

Example 7 Use of Syngenta's Tomato GeneChip to Compare Gene Expressionin Chromosome 2 Between M82 and the Relevant Chromosome 2 Introgressions

An experiment to investigate the profile of gene expression in IL2-3 incomparison to the M82 control was undertaken using the Syngenta TomatoGeneChip. For RNA extraction and tomato GeneChip analysis, flowers weretagged at anthesis before 12.00 noon on consecutive days. Fruit werecollected from plants at: 15 days post anthesis (dpa) (immature green);25 dpa (rapid expansion phase); 40 dpa (mature green); and 54 dpa (redripe). Fruit were flash frozen in liquid nitrogen. Three fruit werepooled to represent one biological replicate. The RNA extractionprotocol was based upon a method using the RNA extraction buffer RNAwiz™(Ambion (Europe) Ltd. UK, Cat #9736) (following the manufacturer'sprotocol). Tomato GeneChip analysis was then undertaken.

The array data was analysed by two-way ANOVA of genotype differences(GeneSpring, Agilent Technologies, UK was used to normalize the data andperform the statistical analysis). A large number of genes showeddifferential expression between M82 and line IL2-3 at 15, 25, 40 and 54days post anthesis. The most apparent changes included elevated levelsof a pectin methylesterase probe ID Le002389 in the IL2-3 lineespecially up to mature green at 40 days post anthesis (FIG. 6).

A total of 29 gene models were tested by RT-PCR against parental linesunder QTL2, and 17 gene models were tested for each of QTLs 4 and 5.

mRNA concentration levels of ethylene responsive transcription factor 12(QTL2), pectin methylesterase (QTL3), dof zinc finger protein 6 (QTL4)and an equilibrative nucleoside transporter family protein (QTL5) weremeasured in M82, IL2-4, IL2-3 and various recombinant lines at themature green and breaker stages. Texture data was also obtained from theouter and inner pericarps of the corresponding fruits.

Ethylene responsive transcription factor 12 was expressed at high levelsin M82 fruit at breaker stage but at low levels in introgression linesand some recombinant lines (FIG. 7). The low expression levels are seenespecially in recombinant lines 301 and 769 which, as shown in FIG. 3,have a higher degree of introgression from S. pennellii than recombinantline 910. Corresponding texture data shows that recombinant lines 301and 769 have firmer fruit than line 910.

Pectin methylesterase was expressed at low levels in M82 fruit but athigh levels in introgression lines and some recombinant lines (FIG. 8).The high expression levels are seen especially in recombinant line 301which, as shown in FIG. 3, has a higher degree of introgression from S.pennellii than recombinant lines 124 and 142. Corresponding texture datashows that recombinant line 301 has firmer fruit than lines 124 and 142.

Dof zinc finger protein 6 was expressed at high levels in M82 fruit butat low levels in introgression lines (FIG. 9). RT-PCR confirmed that afurther candidate gene, an equilibrative nucleoside transporter familyprotein, was differentially expressed between M82 and introgression lineIL2-3 (data not shown).

These results indicate the potential for influencing tomato fruittexture phenotypes using, for example, a GM approach which involvesoverexpressing or silencing the candidate genes above. It would appearthat increased tomato fruit texture corresponds with higher levels ofpectin methylesterase but lower levels of ethylene responsivetranscription factor 12 and dof zinc finger protein 6.

Example 8 Further Resolution of the Mapping Intervals to Less than 0.5Centimorgans by Screening an Additional F2 NIL Population of Approx.4000 Individuals and Anchorage of the QTL to the Physical Map and GenomeSequence

6000 IL2-3×M82F2 individuals were grown under standard greenhouseconditions and screened for recombination events across an intervalwhich included the major and minor QTL and the markers TG451 to TG353(see FIG. 1). Of 6000 F2 lines genotyped, a total of 222 recombinantswere recovered with only 42 additional recombinants falling within themapping interval under the five QTLs. This gave a total of 120 usefulrecombinant individuals that were used to generate a QTL map (FIG. 10,panels A+B).

Example 9 Nomination of Candidate Fruit Firmness-Related Genes

In addition to DNA molecular marker information this invention alsorelates to the identification of putative candidate genes that controlthe described trait. These QTL have been characterised at the molecularlevel and candidate genes at these loci have been identified. Thecurrent list of candidate genes is as follows: QTL1, TG451 a MADS-boxtranscription factor related by sequence to Petunia gene pMADS3, asimilar gene is involved in the regulation of early chloroplastdevelopment in Arabidopsis (Chi et al (2008)). QTL2, ethylene responsivetranscription factor 12 (SL1.00sc00226_365.1). QTL3, apectinesterase/pectinesterase inhibitor (Syngenta GeneChip probe ID,Le0023899).

Where possible, QTL have been anchored to the tomato physical map. Thecandidate sequence for QTL3 is one of 3 putative pectinesterase(pme)/pectinesterase inhibitor genes. The probe on the Syngenta arrayshows homology with all 3 of these pme genes. These genes are so similarin sequence it is difficult to distinguish their individual expressionpatterns even using 3′ end sequence. The three unigenes are SGN-U585819,SGN-U585820, SGN-U58523.

QTL4 candidate gene is the open reading frame corresponding to dof zincfinger protein 6 (SL1.00sc00226_436.1.1) position 4475868 to 4476845 (+strand). QTL5 candidate gene is the open reading frame corresponding toequilibrative nucleoside transporter family protein(SL1.00sc00226_511.1) position 5133572 to 5135660 (− strand).

Example 10 Marker Sequences as Used Herein

TABLE 3  Primers and PCR conditions used to generatemarkers that delineate QTL region on chromosome 2. Annealing TemperatureSEQ Primer Marker (degrees ID Name Primer Sequence Type celcius) 1NT3853F GGATTGTGTTATCTCCGATG SSR 59° C. 2 NT3853R GAAAAAGGGAAGAGATGGGSSR 59° C. 3 NT3907F GAACAGTTTCCGGTGGAG SSR 59° C. 4 NT3907RTTGGGCCAGGAAGAAAAC SSR 59° C. 5 TG14F GCCAACTGATTGCCTATCCT SNP 59° C.marker 6 TG14R CTCACTCCACCCATCACAAC SNP 59° C. marker 7 HOX7AFTGACTCCGGCAAATTTCTCT SNP 59° C. marker 8 HOX7AR TCCCCCATGTATGGACTGAT SNP59° C. marker 9 CT277F TGGTAACCTGCTGTGGTCAA SNP 59° C. marker 10 CT277RAGGTAAACCGCCAGCTCATT SNP 59° C. marker 11 Lm0127F GAAAGGATGCAGCCAAAAATSNP 59° C. marker 12 Lm0127R TACTCTAAGGGCGCACAATG SNP 59° C. marker 13Lm1650F AGGTTGGCAGAAGACGAAGA SNP 59° C. marker 14 Lm1650RACATCCCAAAGAGCTCCAGA SNP 59° C. marker 15 LE5100F TGTTCCAAACGCCTAAAACCSNP 59° C. marker 16 LE5100R TTCCCCAAGTGATTCCTCAG SNP 59° C. marker 17LE5200F TTTGTACAAGCGGCACAAAG SNP 59° C. marker 18 LE5200RCAGCTCGCGTCATCTCATTA SNP 59° C. marker 19 HB2600F AGGGAGGCTGTGGGTAAGATSNP 59° C. marker 20 HB2600R GGCACCTAGACCAAATCCAA SNP 59° C. marker 21TG353F CAGAGCCTGATCTTTCACCA SNP 59° C. marker 22 TG353RTTCGTGTTGGAGATGGAAAG SNP 59° C. marker

TABLE 4 Allele size of Allele size of Method of Marker M82 (bp) LA716(bp) Precision (bp) measurement NT3853 102 96 ±2 sequencer NT3907 192195 ±1 sequencer TG14 381 381 ±1 sequencer HOX7A 257 257 ±1 sequencerCT277 400 450 ±20 Agarose gel Lm0127 223 222 ±1 sequencer Lm1650 252 252±1 sequencer LE5100 278 278 ±1 sequencer LE5200 197 204 ±3 sequencerHB2600 246 246 ±1 sequencer TG353 368 367 ±1 sequencer

What is claimed is:
 1. A Solanum lycopersicum tomato fruit withsignificantly increased fruit firmness at the harvesting stagecomprising at least one genetic element in the cultivated Solanumlycopersicum tomato plant producing said tomato fruit, wherein saidfirmness is measured on the inner pericarp and is from between 1.2 to2.0 times greater than that of fruit from a control Solanum lycopersicumtomato plant which does not have the said at least one genetic element,wherein said genetic element is identical to a genetic element ofSolanum lycopersicum QTL-NIL 301 deposited with NCIMB under accessionnumber 41662, and wherein the said genetic element of QTL-NIL 301 islinked to at least one DNA marker in the cultivated Solanum lycopersicumtomato plant, said at least one DNA marker selected from: a) NT3853,which can be detected by a forward primer of SEQ ID NO: 1 and a reverseprimer of SEQ ID NO: 2; b) NT3907, which can be detected by a forwardprimer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4; c) TG14,which can be detected by a forward primer of SEQ ID NO: 5 and a reverseprimer of SEQ ID NO: 6; d) HOX7A, which can be detected by a forwardprimer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8; e) CT277,which can be detected by a forward primer of SEQ ID NO: 9 and a reverseprimer of SEQ ID NO: 10; f) Lm0127, which can be detected by a forwardprimer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12; g)Lm1650, which can be detected by a forward primer of SEQ ID NO: 13 and areverse primer of SEQ ID NO: 14; h) LE5100, which can be detected by aforward primer of SEQ ID NO: 15 and a reverse primer of SEQ ID NO: 16;i) LE5200, which can be detected by a forward primer of SEQ ID NO: 17and a reverse primer of SEQ ID NO:18; j) HB2600, which can be detectedby a forward primer of SEQ ID NO: 19 and a reverse primer of SEQ ID NO:20; and k) TG353, which can be detected by a forward primer of SEQ IDNO: 21 and a reverse primer of SEQ ID NO:
 22. 2. The tomato fruitaccording to claim 1 wherein the harvesting stage is the mature greenstage.
 3. A cultivated Solanum lycopersicum tomato plant which producesthe tomato fruit according to claim
 1. 4. A Solanum lycopersicum tomatoseed which produces the cultivated tomato plant according to claim
 3. 5.Plant part of the cultivated Solanum lycopersicum tomato plant accordingto claim 3, wherein the plant part comprises the genetic element.
 6. Amethod of producing a cultivated Solanum lycopersicum tomato plant whichprovides fruit with significantly increased fruit firmness bycultivating the tomato seed according to claim
 4. 7. A cultivatedSolanum lycopersicum tomato plant, or part thereof, obtained by themethod according to claim 6, wherein the plant or part thereof comprisesthe genetic element.
 8. A hybrid Solanum lycopersicum tomato plant, orpart thereof, obtained by crossing the cultivated tomato plant accordingto claim 3 with a second Solanum lycopersicum tomato plant, wherein thehybrid tomato plant or part thereof comprises the genetic element. 9.Tomato seed produced by growing the hybrid Solanum lycopersicum tomatoplant of 8, wherein the seed comprises the genetic element.
 10. Tomatoseed produced by crossing the cultivated tomato plant of claim 3 with asecond tomato plant to obtain a seed that produces a plant thatcomprises the genetic element and has significantly increased fruitfirmness compared to a control tomato plant.